Anti-Abeta antibodies and their use

ABSTRACT

The present invention relates to antibody molecules capable of specifically recognizing two regions of the β-A4 peptide, wherein the first region comprises the amino acid sequence AEFRHDSGY as shown in SEQ ID NO: 1 or a fragment thereof and wherein the second region comprises the amino acid sequence VHHQKLVFFAEDVG as shown in SEQ ID NO: 2 or a fragment thereof. Furthermore, nucleic acid molecules encoding the inventive antibody molecules and vectors and hosts comprising said nucleic acid molecules are disclosed. In addition, the present invention provides for compositions, preferably pharmaceutical or diagnostic compositions, comprising the compounds of the invention as well as for specific uses of the antibody molecules, nucleic acid molecules, vectors or hosts of the invention.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. patent application Ser. No.10/505,313, filed Aug. 20, 2004, which is the national stage ofPCT/EP2003/001759, filed Feb. 20, 2003.

FIELD OF THE INVENTION

The present invention relates to antibody molecules capable ofspecifically recognizing two regions of the β-A4 peptide, wherein thefirst region comprises the amino acid sequence AEFRHDSGY as shown in SEQID NO: 1 or a fragment thereof and wherein the second region comprisesthe amino acid sequence VHHQKLVFFAEDVG as shown in SEQ ID NO: 2 or afragment thereof. Furthermore, nucleic acid molecules encoding theinventive antibody molecules and vectors and hosts comprising saidnucleic acid molecules are disclosed. In addition, the present inventionprovides for compositions, preferably pharmaceutical or diagnosticcompositions, comprising the compounds of the invention as well as forspecific uses of the antibody molecules, nucleic acid molecules, vectorsor hosts of the invention.

BACKGROUND OF THE INVENTION

Several documents are cited throughout the text of this specification.Each of the documents cited herein (including any manufacturersspecifications, instructions, etc.) are hereby incorporated byreference.

About 70% of all cases of dementia are due to Alzheimer's disease whichis associated with selective damage of brain regions and neural circuitscritical for cognition. Alzheimer's disease is characterized byneurofibrillary tangles in particular in pyramidal neurons of thehippocampus and numerous amyloid plaques containing mostly a dense coreof amyloid deposits and defused halos.

The extracellular neuritic plaques contain large amounts of apre-dominantly fibrillar peptide termed “amyloid β”, “A-beta”, “A1β4”,“β-A4” or “Aβ”; see Selkoe (1994), Ann. Rev. Cell Biol. 10, 373-403, Koo(1999), PNAS Vol. 96, pp. 9989-9990, U.S. Pat. No. 4,666,829 or Glenner(1984), BBRC 12, 1131. This amyloid is derived from “Alzheimer precursorprotein/β-amyloid precursor protein” (APP). APPs are integral membraneglycoproteins (see Sisodia (1992), PNAS Vol. 89, pp. 6075) and areendoproteolytically cleaved within the Aβ sequence by a plasma membraneprotease, α-secretase (see Sisodia (1992), loc. cit.). Furthermore,further secretase activity, in particular β-secretase and γ-secretaseactivity leads to the extracellular release of amyloid-β (A13)comprising either 39 amino acids (Aβ39), 40 amino acids (Aβ40), 42 aminoacids (Aβ42) or 43 amino acids (Aβ43); see Sinha (1999), PNAS 96,11094-1053; Price (1998), Science 282, 1078 to 1083; WO 00/72880 orHardy (1997), TINS 20, 154.

It is of note that Aβ has several naturally occurring forms, whereby thehuman forms are referred to as the above mentioned Aβ39, Aβ40, Aβ41,Aβ42 and Aβ43. The most prominent form, Aβ42, has the amino acidsequence (starting from the N-terminus):DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 27). In Aβ41,Aβ40, Aβ39, the C-terminal amino acids A, IA and VIA are missing,respectively. In the Aβ43-form an additional threonine residue iscomprised at the C-terminus of the above depicted sequence (SEQ ID NO:27).

The time required to nucleate Aβ40 fibrils was shown to be significantlylonger than that to nucleate Aβ42 fibrils; see Koo, loc. cit. and Harper(1997), Ann. Rev. Biochem. 66, 385-407. As reviewed in Wagner (1999), J.Clin. Invest. 104, 1239-1332, the Aβ42 is more frequently foundassociated with neuritic plaques and is considered to be morefibrillogenic in vitro. It was also suggested that Aβ42 serves as a“seed” in the nucleation-dependent polymerization of orderednon-crystalline Aβ peptides; Jarrett (1993), Cell 93, 1055-1058.

It has to be stressed that modified APP processing and/or the generationof extracellular plaques containing proteinaceous depositions are notonly known from Alzheimer's pathology but also from subjects sufferingfrom other neurological and/or neurodegenerative disorders. Thesedisorders comprise, inter alia, Down's syndrome, Hereditary cerebralhemorrhage with amyloidosis Dutch type, Parkinson's disease, ALS(amyotrophic lateral sclerosis), Creutzfeld Jacob disease, HIV-relateddementia and motor neuropathy.

In order to prevent, treat and/or ameliorate disorders and/or diseasesrelated to the pathological deposition of amyloid plaques, means andmethods have to be developed which either interfere with β-amyloidplaque formation, which are capable of preventing Aβ aggregation and/orare useful in de-polymerization of already formed amyloid deposits oramyloid-β aggregates.

Accordingly, and considering the severe defects of modified and/orpathological amyloid biology, means and methods for treating amyloidrelated disorders are highly desirable. In particular, efficient drugswhich either interfere with pathological amyloid aggregation or whichare capable of de-polymerization of aggregated Aβ are desired.Furthermore, diagnostic means are desirable to detect, inter alia,amyloid plaques.

Thus, the technical problem of the present invention is to comply withthe needs described herein above.

BRIEF SUMMARY OF THE INVENTION

Accordingly, the present invention relates to an antibody moleculecapable of specifically recognizing two regions of the β-A4/Aβ4 peptide,wherein the first region comprises the amino acid sequence AEFRHDSGY(SEQ ID NO: 1) or a fragment thereof and wherein the second regioncomprises the amino acid sequence VHHQKLVFFAEDVG (SEQ ID NO: 2) or afragment thereof.

In context of the present invention, the term “antibody molecule”relates to full immunoglobulin molecules, preferably IgMs, IgDs, IgEs,IgAs or IgGs, more preferably IgG1, IgG2a, IgG2b, IgG3 or IgG4 as wellas to parts of such immunoglobulin molecules, like Fab-fragments orV_(L)-, V_(H)- or CDR-regions. Furthermore, the term relates to modifiedand/or altered antibody molecules, like chimeric and humanizedantibodies. The term also relates to modified or altered monoclonal orpolyclonal antibodies as well as to recombinantly or syntheticallygenerated/synthesized antibodies. The term also relates to intactantibodies as well as to antibody fragments/parts thereof, like,separated light and heavy chains, Fab, Fab/c, Fv, Fab′, F(ab′)₂. Theterm “antibody molecule” also comprises antibody derivatives, thebifunctional antibodies and antibody constructs, like single chain Fvs(scFv), bispecific scFvs or antibody-fusion proteins. Further details onthe term “antibody molecule” of the invention are provided herein below.

The term “specifically recognizing” means in accordance with thisinvention that the antibody molecule is capable of specificallyinteracting with and/or binding to at least two amino acids of each ofthe two regions of β-A4 as defined herein. Said term relates to thespecificity of the antibody molecule, i.e. to its ability todiscriminate between the specific regions of the β-A4 peptide as definedherein and another, not related region of the β-A4 peptide or another,not APP-related protein/peptide/(unrelated) tests-peptide. Accordingly,specificity can be determined experimentally by methods known in the artand methods as disclosed and described herein. Such methods comprise,but are not limited to Western blots, ELISA-, RIA-, ECL-, IRMA-tests andpeptide scans. Such methods also comprise the determination ofK_(D)-values as, inter alia, illustrated in the appended examples. Thepeptide scan (pepspot assay) is routinely employed to map linearepitopes in a polypeptide antigen. The primary sequence of thepolypeptide is synthesized successively on activated cellulose withpeptides overlapping one another. The recognition of certain peptides bythe antibody to be tested for its ability to detect or recognize aspecific antigen/epitope is scored by routine colour development(secondary antibody with horseradish peroxidase and 4-chloronaphthol andhydrogenperoxide), by a chemoluminescence reaction or similar meansknown in the art. In the case of, inter alia, chemoluminescencereactions, the reaction can be quantified. If the antibody reacts with acertain set of overlapping peptides one can deduce the minimum sequenceof amino acids that are necessary for reaction; see illustrative Example6 and appended Table 2.

The same assay can reveal two distant clusters of reactive peptides,which indicate the recognition of a discontinuous, i.e. conformationalepitope in the antigenic polypeptide (Geysen (1986), Mol. Immunol. 23,709-715).

In addition to the pepspot assay, standard ELISA assay can be carriedout. As demonstrated in the appended examples small hexapeptides may becoupled to a protein and coated to an immunoplate and reacted withantibodies to be tested. The scoring may be carried out by standardcolour development (e.g. secondary antibody with horseradish peroxidaseand tetramethyl benzidine with hydrogenperoxide). The reaction incertain wells is scored by the optical density, for example at 450 nm.Typical background (=negative reaction) may be 0.1 OD, typical positivereaction may be 1 OD. This means the difference (ratio)positive/negative can be more than 10 fold. Further details are given inthe appended examples. Additional, quantitative methods for determiningthe specificity and the ability of “specifically recognizing” the hereindefined two regions of the β-A4 peptide are given herein below.

The term “two regions of the β-A4 peptide” relates to two regions asdefined by their amino acid sequences shown in SEQ ID NOs: 1 and 2,relating to the N-terminal amino acids 2 to 10 and to the central aminoacids 12 to 25 of β-A4 peptide. The term “β-A4 peptide” in context ofthis invention relates to the herein above described Aβ39, Aβ41, Aβ43,preferably to Aβ40 and Aβ42. Aβ42 is also depicted in appended SEQ IDNO: 27. It is of note that the term “two regions of the β-A4 peptide”also relates to an “epitope” and/or an “antigenic determinant” whichcomprises the herein defined two regions of the β-A4 peptide or partsthereof. In accordance with this invention, said two regions of the β-A4peptide are separated (on the level of the amino acid sequence) in theprimary structure of the β-A4 peptide by at least one amino acid,preferably by at least two amino acids, more preferably by at leastthree amino acids, more preferably by at least four amino acids, morepreferably by at least five amino acids, more preferably at least sixamino acids, more preferably at least nine amino acids and mostpreferably at least twelve amino acids. As shown herein and asdocumented in the appended examples, the inventive antibodies/antibodymolecules detect/interact with and/or bind to two regions of the β-A4peptide as defined herein, whereby said two regions are separated (onthe primary structure level of the amino acid sequence) by at least oneamino acid and wherein the sequence separating said tworegions/“epitope” may comprise more then ten amino acids, preferably 14amino acids, more preferably 15 amino acids or 16 amino acids. Forexample, MSR-3 Fab (as an inventive antibody molecule) recognizesdetects/interacts with two regions on the β-A4 peptide, wherein saidfirst region comprises amino acids 3 and 4 (EF) and said second regionscomprises amino acids 18 to 23 (VFFAED, SEQ ID NO: 421). Accordingly,the separating sequence between the region/epitopes to bedetected/recognized has a length of 13 amino acids on the primary aminoacid sequence structure. Similarly, MSR #3.4H7 IgG1, an optimized andmatured antibody molecules derived from MSR-3 and comprised in anIgG1-framework, detects/interacts with/binds to two epitopes/regions ofβ-A4 which comprise in the first region positions 1 to 4 (DAEF) and inthe second region positions 19 to 24 (FFAEDV, SEQ ID NO: 423) of 8-A4 asdefined herein. Accordingly, MSR #3.4H7 IgG1recognizes/detects/interacts with/binds to two epitopes/regions whichare, on the primary amino acid sequence level, separated by 14 aminoacids. As detailed in the appended examples, affinity maturation andconversion of monovalent inventive Fab fragments to full-length IgG1antibodies may result in a certain broadening of the epitopes/regionsdetected in pepspot, ELISA assays and the like. Therefore, the antibodymolecules of the invention are capable of simultaneously andindependently recognizing two regions of the β-A4 peptide/Aβ4 whereinsaid regions comprise the amino acid sequence as shown in SEQ ID NO: 1(or parts thereof) and the amino acid sequence as shown in SEQ ID NO: 2(or (a) part(s) thereof). Due to the potential broadening of epitopes asdetailed herein it is, however, also envisaged that amino acids in closeproximity to the sequences of SEQ ID NO: 1 and 2 aredetected/recognized, i.e. that additional amino acids are part of thetwo regions to be detected/recognized. Accordingly, it is also envisagedthat, e.g. the first amino acid of Aβ (1-42) as defined herein, namely D(Aspartic acid) in part of one epitope to be detected/recognized or thatamino acids located after the region of Aβ (1-42) as defined in SEQ IDNO: 2 are detected/recognized. Said additional amino acid may, e.g., bethe amino acid on position 26 of SEQ ID NO: 27 (βA4/Aβ (1-42)), namely S(Serine).

The term may also relate to a conformational epitope, a structuralepitope or a discountinuous epitope consisting of said two regions orparts thereof; see also Geysen (1986), loc. cit. In context of thisinvention, a conformational epitope is defined by two or more discreteamino acid sequences separated in the primary sequence which cometogether on the surface when the polypeptide folds to the native protein(Sela, (1969) Science 166, 1365 and Layer, (1990) Cell 61, 553-6). Theantibody molecules of the present invention are envisaged tospecifically bind to/interact with a conformational/structuralepitope(s) composed of and/or comprising the two regions of β-A4described herein or parts thereof as disclosed herein below. The“antibody molecules” of the present invention are thought to comprise asimultaneous and independent dual specificity to (a) an amino acidstretch comprising amino acids 2 to 10 (or (a) part(s) thereof) of β-A4and (b) an amino acid stretch comprising amino acids 12 to 25 (or (a)part(s) thereof) of β-A4 (SEQ ID NO. 27). Fragments or parts of thesestretches comprise at least two, more preferably at least three aminoacids. Preferred fragments or parts are in the first region/stretch ofSEQ ID NO: 27 the amino acid sequences AEFRHD (SEQ ID NO: 415), EF, EFR,FR, EFRHDSG (SEQ ID NO: 416), EFRHD (SEQ ID NO: 417) or HDSG (SEQ ID NO:418), and in the second region/stretch of SEQ ID NO: 27 the amino acidsequences HHQKL (SEQ ID NO: 419), LV, LVFFAE (SEQ ID NO: 420), VFFAED(SEQ ID NO: 421), VFFA (SEQ ID NO: 422) or FFAEDV (SEQ ID NO: 423). Asmentioned above, said fragments may also comprise additional amino acidsor may be parts of the fragments defined herein. Specific examples areDAE, DAEF, FRH or RHDSG.

DETAILED DESCRIPTION OF THE INVENTION

A number of antibodies specifically recognizing Aβ peptides have beendescribed in the art. These antibodies have mainly been obtained byimmunizing animals with Aβ1-40 or Aβ1-42 or fragments thereof usingstandard technologies. According to published data monoclonal antibodiesthat were generated by immunization with the complete Aβ peptide (1-40or 1-42) recognize exclusively an epitope close to the N-terminus of Aβ.Further, examples are the antibodies BAP-1 and BAP-2 (Brockhaus,unpublished) which were generated by immunization of mice with Aβ1-40and which recognize the amino acids 4-6 in the context of larger Aβpeptides; see appended Example 7, Table 2 and Example 12, Table 7.Antibodies that recognize the middle part of Aβ derive fromimmunizations with smaller peptides. For example, the antibody 4G8 wasgenerated by immunization with the AR peptide 1-24 and recognizesexclusively the sequence 17-24 (Kim, (1988) Neuroscience ResearchCommunications 2, 121-130). Many other monoclonal antibodies have beengenerated by immunizing mice with A8-derived fragments, and antibodiesrecognizing the C-terminal end of Aβ1-40 and Aβ1-42 are widely used todistinguish and quantitate the corresponding Aβ peptides in biologicalfluids and tissues by ELISA, Western blot and immunohistochemistryanalysis (Ida et al, (1996) J. Biol. Chem. 271, 22908-22914;Johnson-Wood et al., (1997), Proc. Natl. Acad. Sci. USA (1994),1550-1555; Suzuki et al., (1994), Science 264, 1336-1340; Brockhaus(1998), Neuro Rep. 9, 1481-1486). BAP-17 is a mouse monoclonal antibodywhich has been generated by immunizing mice with Aβ fragment 35-40. Itspecifically recognizes the C-terminal end of Aβ1-40 (Brockhaus (1998)Neuroreport 9, 1481-1486).

It is believed that the immunization with T-cell dependent antigens(often poor immunogens) requires a proteolytic cleavage of the antigenin the endosomes of antigen presenting cells. The in vivo selection ofhigh affinity antibodies after immunization is driven by the contact ofhelper T cells to antigen presenting cells. The antigen presenting cellsonly present short peptides and not polypeptides of large size.Accordingly, these cells have a complicated (but well known) machineryto endocytose antigen(s), degrade the antigen(s) in endosomes, combineselected peptides with suitable MHC class II molecules, and to exportthe peptide-MHC complex to the cell surface. This is where the antigenspecific recognition by T cells occurs, with the aim to provide help tomaturing B cells. The B cells which receive most T cell help have thebest chance to develop into antibody secreting cells and to proliferate.This shows that antigen processing by proteolysis is an important stepfor the generation of an high affinity antibody response in vivo and mayexplain the dominance of the N-terminal Aβ epitope in prior artmonoclonal and polyclonal antibodies derived by immunization.

In contrast, the selection of antibodies/antibody molecules of thepresent invention is driven by the physical adherence of Fab expressingphages to the antigen. There is no degradation of the antigen involvedin this in vitro selection process. The phages which express the Fabwith the highest affinity towards the antigen are selected andpropagated. A synthetic library as employed in the appended examples toselect for specific antibody molecules according to this invention isparticularly suited for avoiding any bias for single, continuousepitopes that is often found in libraries derived from immunized Bcells.

It is of note that the prior art has not described antibody moleculesrecognizing two, independent regions of Aβ4 which specificallyrecognizes (a) discontinuous/structural/conformational epitope(s) and/orwhich are capable of simultaneously and independently recognizing tworegions/epitopes of Aβ4. Vaccination of transgenic mice overexpressingmutant human APP_(V717F) (PDAPP mice) with Aβ1-42 resulted in an almostcomplete prevention of amyloid deposition in the brain when treatmentwas initiated in young animals, i.e. before the onset ofneuropathologies, whereas in older animals a reduction of already formedplaques was observed suggesting antibody-mediated clearance of plaques(Schenk et al., (1999), Nature 400, 173-177). The antibodies generatedby this immunization procedure were reactive against the N-terminus ofAβ4 covering an epitope around amino acids 3-7 (Schenk et al., (1999),loc. cit.; WO 00/72880). Active immunization with Aβ1-42 also reducedbehavioural impairment and memory loss in different transgenic modelsfor Alzheimer's Disease (Janus et al., (2000) Nature 408, 979-982;Morgan et al., (2000) Nature 408, 982-985). Subsequent studies withperipherally administered antibodies, i.e. passive immunization, haveconfirmed that antibodies can enter the central nervous system, decorateplaques and induce clearance of preexisting amyloid plaques in APPtransgenic mice (PDAPP mice) (Bard et al., (2000) Nat. Med. 6, 916-919;WO 00/72880). In these studies, the monoclonal antibodies with the mostpotent in vivo and ex vivo efficacy (triggering of phagocytosis inexogenous microglial cells) were those which recognized Aβ4 N-terminalepitopes 1-5 (mab 3D6, IgG2b) or 3-6 (mab 10D5, IgG1). Likewise,polyclonal antibodies isolated from mice, rabbits or monkeys afterimmunization with Aβ1-42 displayed a similar N-terminal epitopespecificity and were also efficacious in triggering phagocytosis and invivo plaque clearing. In contrast, C-terminal specific antibodiesbinding to Aβ1-40 or Aβ1-42 with high affinity did not inducephagocytosis in the ex vivo assay and were not efficacious in vivo (WO00/72880). Monoclonal antibody m266 (WO 00/72880) was raised againstAβ13-28 (central domain of Aβ) and epitope mapping confirmed theantibody specificity to cover amino acids 16-24 in the Aβ sequence. Thisantibody does not bind well to aggregated Aβ and amyloid deposits andmerely reacts with soluble (monomeric) Aβ, i.e. properties which aresimilar to another well-known and commercially available monoclonalantibody (4G8; Kim, (1988) Neuroscience Research Communications 2,121-130; commercially available from Signet Laboratories Inc. Dedham,Mass. USA) which recognizes the same epitope.

In vivo, the m266 antibody was recently found to markedly reduce Aβdeposition in PDAPP mice after peripheral administration (DeMattos,(2001) Proc. Natl. Acad. Sci. USA 98, 8850-8855). However, and incontrast to N-terminal specific antibodies, m266 did not decorateamyloid plaques in vivo, and it was therefore hypothesized that thebrain Aβ burden was reduced by an antibody-induced shift in equilibriumbetween CNS and plasma Aβ resulting in the accumulation of brain-derivedAβ in the periphery, firmly complexed to m266 (DeMattos, (2001) loc.cit.).

The antibodies/antibody molecules of the present invention, bysimultaneously (for example in a structural/conformational epitopeformed by the N-terminal and central region of βA4 as described herein)and independently (for example in pepspot assays as documented in theappended experimental part) binding to the N-terminal and centralepitopes, combine the properties of an N-terminal-specific antibody anda central epitope-specific antibody in a single molecule. Antibodieswith the dual epitope specificity, as described in the presentinvention, are considered to be more efficacious in vivo, in particularin medical and diagnostic settings for, e.g., reducing amyloid plaqueburden or amyloidogenesis or for the detection of amyloid deposits andplaques. It is well known that in the process of Aβ4 aggregation andamyloid deposition conformational changes occur, and while the centralepitope is easily accessible in soluble Aβ4 it appears to be hidden andless reactive in aggregated or fibrillar Aβ4. The fact that thecentral/middle epitope-specific antibody m266 is efficacious in vivoindicates that neutralization of soluble Aβ4 may also be a criticalparameter. The antibodies/antibody molecules of the present invention,due to the dual epitope specificity, can bind to both fibrillar andsoluble Aβ4 with similar efficacy, thus allowing interaction withamyloid plaques as well as neutralization of soluble Aβ4. The term“simultaneously and independently binding to the N-terminal andcentral/middle epitopes of β-A4” as employed herein in context of theinventive antibody molecules relates to the fact that theantibodies/antibody molecules described herein may detect and/or bind toboth epitopes simultaneously, i.e. at the same time (for example onconformational/structural epitopes formed by the N-terminal epitope (or(a) part(s) thereof) and central epitopes (or (a) part(s) thereof) ofβA4 as defined herein) and that the same antibody molecules, however,are also capable of detecting/binding to each of the defined epitopes inan independent fashion, as inter alia, demonstrated in the pepspotanalysis shown in the examples.

Clearance of amyloid plaques in vivo in PDAPP mice after directapplication of the antibodies to the brain is not dependent on the IgGsubtype and may also involve a mechanism which is not Fc-mediated, i.e.no involvement of activated microglia in plaque clearance (Bacskai,(2001), Abstract Society for Neuroscience 31^(st) Annual Meeting, Nov.10-15, 2001, San Diego). This observation is in contrast to what hasbeen postulated in an earlier study by Bard (2000), loc. cit.

In another study antibodies raised against Aβ1-28 and Aβ1-16 peptideswere found to be effective in disaggregating Aβ fibrils in vitro,whereas an antibody specific for Aβ13-28 was much less active in thisassay (Solomon, (1997) Proc. Natl. Acad. Sci. USA 94, 4109-4112).Prevention of Aβ aggregation by an anti-Aβ1-28 antibody (AMY-33) hasalso been reported (Solomon, (1996) Proc. Natl. Acad. Sci. USA 93,452-455). In the same study, antibody 6F/3D which has been raisedagainst Aβ fragment 8-17 slightly interfered with Zn²⁺-induced Aβaggregation but had no effect on the self aggregation induced by otheraggregation-inducing agents.

The efficacy of the various antibodies in these in vitro assayscorrelates with the accessibility of their epitopes in Aβ4 aggregates.The N-terminus is exposed and N-terminal specific antibodies clearlyinduce de-polymerization, whereas the central region and the C-terminusare hidden and not easily accessible and thus antibodies against theseepitope are much less effective.

Investigations with respect to epitope accessibilty for antibodies haveshown that in aggregated Aβ the N-terminal epitope is exposed and reactswith the BAP-1 antibody, whereas the middle or central epitope indeedremains cryptic, i.e. no binding of the 4G8 antibody was observed.However, in monomeric Aβ both epitopes are overt and are equallyrecognized by both prior art antibodies.

In contrast, in the present invention, it was surprisingly found thatthe herein described antibody molecules recognize two discontinuousamino acid sequences, e.g. a conformational/structural epitope on the Aβpeptide. Two “discontinuous amino acid sequences” in accordance withthis invention means that said two amino acid sequences forming theN-terminal and central/middle epitopes, respectively, are separated onβ-A4 in its primary structure by at least two amino acids which are notpart of either epitope.

The binding area of an antibody Fab (=paratope) occupies a molecularsurface of approximately 30×30 Å in size (Layer, Cell 61 (1990),553-556). This is enough to contact 15 to 22 amino acid residues whichmay be present on several surface loops. The discontinuous epitoperecognized by the inventive antibody molecules resembles a conformationin which the N-terminal (residues 2 to 10 or parts thereof) and middleAβ peptide sequences (residues 12 to 25 or parts thereof) are in closeproximity. Only within this conformation, the maximum number ofantigen-antibody contacts and the lowest free energy state are obtained.

Based on energetic calculations it has been suggested that a smallersubset of 5-6 residues, which are not arranged in a linear sequence butare scattered over the epitope surface, contributes most of the bindingenergy while surrounding residues may merely constitute a complementaryarray (layer (1990) loc. cit.).

The inventive antibodies/antibody molecules are capable of binding toaggregated Aβ and strongly react with amyloid plaques in the brain of ADpatients (as documented in the appended examples). In addition, they arecapable of de-polymerizing/disintegrating amyloid aggregates.

Without being bound by theory, the conformational/structural epitope(composed by the two regions of Aβ4 or (a) part(s) of said regions asdescribed herein) is believed to be partially exposed in aggregated Aβ.However, it is known that major part of the middle/second epitope/regionalone is not freely accessible in these Aβ aggregates (based on the poorreactivities of middle epitope-specific antibodies 4G8 and m266). On theother hand, and in view of the considerations mentioned above, it islikely that one or several residues of the middle region are componentsof the conformational epitope and, in conjunction with the residues fromthe N-terminal region, are accessible to the antibodies of the presentinvention, thereby significantly contributing to the binding energy ofthe antibody-Aβ4 interaction. The reactivity of the inventive antibodymolecules with the conformational epitope in aggregated Aβ is thereforeunique and clearly distinct from α-Aβ4 antibodies described in the priorart. Yet, as pointed out herein above, a further unique feature of theinventive antibodies/antibody molecules is their capacity tosimultaneously and independently binding to/recognizing two separateepitopes on β-A4, as defined herein and in the appended examples.

In a preferred embodiment of the invention, the inventive antibodymolecule is an antibody molecule wherein the least two regions of theβ-A4 to be specifically recognized by said antibody form aconformational/structural epitope or a discontinuous epitope; see Geysen(1986), loc. cit.; Ghoshal (2001), J. Neurochem. 77, 1372-1385;Hochleitner (2000), J. 1 mm. 164, 4156-4161; layer (1990), loc. cit. Theterm “discontinuous epitope” means in context of the inventionnon-linear epitopes that are assembled from residues from distantportions of the polypeptide chain. These residues come together on thesurface when the polypeptide chain folds into a three-dimensionalstructure to constitute a conformational/structural epitope. The presentinvention provides for preferred, unexpected epitopes within β-A4, whichresult in the inventive generation of specific antibody molecules,capable of specifically interacting with these epitopes. These inventiveantibodies/antibody molecules provide the basis for increased efficacy,and a reduced potential for side effects. As pointed out above, theinventive antibodies, however, were also capable of independentlyinteracting with each of the defined two regions/epitopes of β-A4, forexample in Pepspot assays as documented in the appended examples.

The present invention, accordingly, provides for unique tools which maybe employed to de-polymerize aggregated A6-fibrils in vivo and in vitroand/or which are capable of stabilizing and/or neutralizing aconformational epitope of monomeric Aβ and thereby capable of preventingthe pathological Aβ aggregation.

It is furthermore envisaged that the inventive antibodies bind to Aβdeposits at the rim of amyloid plaques in, inter alia, Alzheimer's brainand efficiently dissolve the pathological protofibrils and fibrils.

In a preferred embodiment, the antibody molecule of the inventionrecognizes at least two consecutive amino acids within the two regionsof Aβ4 defined herein, more preferably said antibody molecule recognizesin the first region an amino acid sequence comprising the amino acids:AEFRHD (SEQ ID NO: 415), EF, EFR, FR, EFRHDSG (SEQ ID NO: 416), EFRHD(SEQ ID NO: 417) or HDSG (SEQ ID NO: 418), and in the second region anamino acid sequence comprising the amino acids: HHQKL (SEQ ID NO: 419),LV, LVFFAE (SEQ ID NO: 420), VFFAED (SEQ ID NO: 421), VFFA (SEQ ID NO:422) or FFAEDV (SEQ ID NO: 423). Further fragments or broadened partscomprise: DAE, DAEF, FRH or RHDSG.

It is particularly preferred that the antibody molecule of the inventioncomprises a variable V_(H)-region as encoded by a nucleic acid moleculeas shown in SEQ ID NO: 3, 5 or 7 or a variable V_(H)-region as shown inthe amino acid sequences depicted in SEQ ID NOs: 4, 6 or 8. Thesequences as shown in SEQ ID NOs: 3 and 4 depict the coding region andthe amino acid sequence, respectively, of the V_(H)-region of theinventive, parental antibody MSR-3 (MS-Roche 3), the sequences in SEQ IDNOs: 5 and 6 depict the coding region and the amino acid sequence,respectively, of the V_(H)-region of the inventive, parental antibodyMSR-7 (MS-Roche 7) and SEQ ID NOs: 7 and 8 depict the coding region andthe amino acid sequence, respectively, of the V_(H)-region of theinventive, parental antibody MSR-8 (MS-Roche 8). Accordingly, theinvention also provides for antibody molecules which comprise a variableV_(L)-region as encoded by a nucleic acid molecule as shown in a SEQ IDNO selected from the group consisting of SEQ ID NO: 9, 11 or 13 or avariable V_(L)-region as shown in the amino acid sequences depicted inSEQ ID NOs: 10, 12 or 14. SEQ ID NOs: 9 and 10 correspond to theV_(L)-region of MSR-3, SEQ ID NOs: 11 and 12 correspond to theV_(L)-region of MSR-7 and SEQ ID NOs: 13 and 14 correspond to theV_(L)-region of MSR-8. As illustrated in the appended examples, theparental antibodies MSR-3, -7 and -8, are employed to further generateoptimized antibody molecules with even better properties and/or bindingaffinities. Some of the corresponding and possible strategies areexemplified and shown in the appended examples.

The optimization strategy as illustrated in the appended examples leadto a plurality of inventive, optimized antibodies. These optimizedantibodies share with their parental antibodies the CDR-3 domain of theV_(H)-region. Whereas the original framework region (as shown inappended FIG. 1) remains the same, in the matured/optimized antibodymolecules, CDR1, CDR2 and/or V_(L) CDR3-regions are changed.Illustrative, modified sequence motives for optimized antibody moleculesare shown in appended table 1. Accordingly, within the scope of thepresent invention are also optimized antibody molecules which arederived from the herein disclosed MSR-3, -7 and -8 and which are capableof specifically reacting with/specifically recognizing the two regionsof the β-A4 peptide as defined herein. In particular, CDR-regions,preferably CDR1s, more preferably CDR1s and CDR2s, most preferablyCDR1s, CDR2s and CDR3s as defined herein may be employed to generatefurther inventive antibodies/antibody molecules, inter alia, byCDR-grafting methods known in the art; see Jones (1986), Nature 321,522-515 or Riechmann (1988), Nature 332, 323-327. Most preferably theinventive antibodies/antibody molecules as well as antibody fragments orderivatives are derived from the parental antibodies as disclosed hereinand share, as disclosed above, the CDR-3 domain of the V_(H)-region withat least one of said parental antibodies. As illustrated below, it isalso envisaged that cross-cloned antibodies are generated which are tobe considered as optimized/maturated antibodies/antibody molecules ofthe present invention. Accordingly, preferred antibody molecules mayalso comprise or may also be derived from antibodies/antibody moleculeswhich are characterized by V_(H)-regions as shown in any of SEQ ID NOs:32 to 45 or V_(L)-regions as shown in SEQ ID NOs: 46 to 59 or which maycomprise a CDR-3 region as defined in any of SEQ ID NOs: 60 to 87. In aparticular preferred embodiment, the optimized antibody molecule of thepresent invention comprises V_(H)-regions and V_(L)-regions as depictedin SEQ ID NOs: 88/89 and 90/91, respectively, or parts thereof. Apartthereof may be (a) CDR-region(s), preferably (a) CDR3-region(s). Aparticularly preferred antibody molecule of the optimized type comprisesa H-CDR3 as characterized in SEQ ID NOs: 92 or 93 and/or a L-CDR3 ascharacterized in SEQ ID NOs: 94 or 95.

It is preferred that the antibodies/antibody molecules of the inventionare characterized by their specific reactivity with β-A4 and/or peptidesderived from said β-A4. For example, optical densities in ELISA-tests,as illustrated in the appended examples, may be established and theratio of optical densities may be employed to define the specificreactivity of the parental or the optimized antibodies. Accordingly, apreferred antibody of the invention is an antibody which reacts in anELISA-test with β-A4 to arrive at an optical density measured at 450 nmthat is 10 times higher than the optical density measured without β-A4,i.e. 10 times over background. Preferably the measurement of the opticaldensity is performed a few minutes (e.g. 1, 2, 3, 4, 5, 6, or 7 minutes)after initiation of the color developing reaction in order to optimizesignal to background ratio.

In a particular preferred embodiment, the inventive antibody moleculecomprises at least one CDR3 of an V_(L)-region as encoded by a nucleicacid molecule as shown in SEQ ID NOs: 15, 17 or 19 or at least one CDR3amino acid sequence of an V_(L)-region as shown in SEQ ID NOs: 16, 18 or20 and/or said antibody molecule comprises at least one CDR3 of anV_(H)-region as encoded by a nucleic acid molecule as shown in SEQ IDNOs: 21, 23 or 25 or at least one CDR3 amino acid sequence of anV_(H)-region as shown in SEQ ID NOs: 22, 24 or 26. Most preferred areantibodies comprising at least one CDR3 of an V_(H)-region as definedherein. The CDR-3 domains mentioned herein above relate to theinventive, illustrative parental antibody molecules MSR-3, -7, or -8.However, as illustrated in the appended tables 1, 8 or 10, maturedand/or optimized antibody molecules obtainable by the methods disclosedin the appended examples may comprise modified V_(H)-, CDR1, CDR2 andCDR3 regions. Accordingly, the antibody molecule of the invention ispreferably selected from the group consisting of MSR-3, -7 and -8 or anaffinity-matured version of MSR-3, -7 or -8. Affinity-matured as well ascross-cloned versions of MSR-3, -7 and -8 comprise, inter alia, antibodymolecules comprising CDR1, CDR2 and/or CDR3 regions as shown in table 1or 8 or characterized in any of SEQ ID NOs: 15 to 20, 21 to 26, 60 to74, 75 to 87, 92 and 93 or 94 and 95 as well as in SEQ ID NOs: 354 to413. Most preferably, the antibody of the invention comprises at leastone CDR, preferably a CDR1, more preferably a CDR2, most preferably aCDR3 as shown in the appended table 1, 8 or as documented in appendedtable 10.

It is of note that affinity-maturation techniques are known in the art,described in the appended examples and, inter alia, in Knappik (2000),J. Mol. Biol. 296, 55; Krebs (2000), J. 1 mm. Meth. 254, 67-84; WO01/87337; WO 01/87338; U.S. Pat. No. 6,300,064; EP 96 92 92 78.8 andfurther references cited herein below.

In a more preferred embodiment of the invention, the antibody moleculeis a full antibody (immunoglobulin, like an IgG1, an IgG2, an IgG2b, anIgG3, an IgG4, an IgA, an IgM, an IgD or an IgE), an F(ab)-, Fabc-, Fv-,Fab′-, F(ab′)₂-fragment, a single-chain antibody, a chimeric antibody, aCDR-grafted antibody, a bivalent antibody-construct, an antibody-fusionprotein, a cross-cloned antibody or a synthetic antibody. Also envisagedare genetic variants of immunoglobulin genes. Genetic variants of, e.g.,immunoglobulin heavy G chain subclass 1 (IgG1) may comprise the G1m(17)or G1m(3) allotypic markers in the CH1 domain, or the G1m(1) or theG1m(non-1) allotypic marker in the CH3 domain. The antibody molecule ofthe invention also comprises modified or mutant antibodies, like mutantIgG with enhanced or attenuated Fc-receptor binding or complementactivation. It is also envisaged that the antibodies of the inventionare produced by conventional means, e.g. the production of specificmonoclonal antibodies generated by immunization of mammals, preferablymice, with peptides comprising the two regions of βA4 as defined herein,e.g. the N-terminal and central region/epitope comprising (a) aminoacids 2 to 10 (or (a) part(s) thereof) of β-A4 and (b) an amino acidstretch comprising amino acids 12 to 25 (or (a) part(s) thereof) of β-A4(SEQ ID NO. 27). Accordingly, the person skilled in the art may generatemonoclonal antibodies against such a peptide and may screen the obtainedantibodies for the capacity to simultaneously and independently bindingto/reacting with the N-terminal and central region/epitope of βA4 asdefined herein. Corresponding screening methods are disclosed in theappended examples.

As illustrated in the appended examples, the inventiveantibodies/antibody molecules can readily and preferably berecombinantly constructed and expressed. Preferably, the antibodymolecule of the invention comprises at least one, more preferably atleast two, preferably at least three, more preferably at least four,more preferably at least five and most preferably six CDRs of the hereindefined MSR-3, MSR-7 or MSR-8 parental antibodies or ofaffinity-matured/optimized antibodies derived from said parentalantibodies. It is of note that also more than six CDRs may be comprisedin recombinantly produced antibodies of the invention. The personskilled in the art can readily employ the information given in theappended examples to deduce corresponding CDRs of the parental as wellas the affinity optimized antibodies. Examples of optimized antibodieswhich have been obtained by maturation/optimization of the parentalantibodies are, inter alia, shown in appended table 1. Anmaturated/optimized antibody molecule of the invention is, e.g. MSR7.9H7 which is also characterized by sequences appended herein, whichcomprise SEQ ID NOs: 88 to 95 and which depict the V_(H)-region of MSR7.9H7 (SEQ ID NOs: 88 and 89), the V_(L)-region of MSR 7.9H7 (SEQ IDNOs: 90 and 91), the H-CDR3 of MSR 7.9H7 (SEQ ID NOs: 92 and 93) as wellas the L-CDR3 of MSR 7.9H7 (SEQ ID NOs: 94 and 95). Illustrativeantibody molecule 7.9H7 is derived from parental antibody MSR7 and is aparticular preferred inventive example of an optimized/matured antibodymolecule of the present invention. This antibody molecule may be furthermodified in accordance with this invention, for example in form ofcross-cloning, see herein below and appended examples.

As documented in the appended examples, the antibodies of the inventionalso comprise cross-cloned antibodies, i.e. antibodies comprisingdifferent antibody regions (e.g. CDR-regions) from one or more parentalor affinity-optimized antibody(ies) as described herein.

These cross-cloned antibodies may be antibodies in several, differentframeworks, whereby the most preferred framework is an IgG-framework,even more preferred in an IgG1-, IgG2a or an IgG2b-framework. It isparticularly preferred that said antibody framework is a mammalian, mostpreferably a human framework. The domains on the light and heavy chainshave the same general structure and each domain comprises four frameworkregions, whose sequences are relatively conserved, joined by threehypervariable domains known as complementarity determining regions(CDR1-3).

As used herein, a “human framework region” relates to a framework regionthat is substantially identical (about 85% or more, usually 90-95% ormore) to the framework region of a naturally occurring humanimmunoglobulin. The framework region of an antibody, that is thecombined framework regions of the constituent light and heavy chains,serves to position and align the CDR's. The CDR's are primarilyresponsible for binding to an epitope of an antigen. It is of note thatnot only cross-cloned antibodies described herein may be presented in apreferred (human) antibody framework, but also antibody moleculescomprising CDRs from, inter alia, the parental antibodies MSR-3, -7 or-8 as described herein or of matured antibodies derived from saidparental antibodies, may be introduced in an immunoglobulin framework.Preferred frameworks are IgG1, IgG2a and IgG2b. Most preferred are humanframeworks and human IgG1 frameworks.

As shown in the appended examples, it is, inter alia possible, totransfer, by genetic engineering known in the art whole light chainsfrom an optimized donor clone to an optimized recipient clone. Examplefor an optimized donor clone is, e.g. L-CDR1 (L1) and an example for anoptimized recipient clone is H-CDR2 (H2). Epitope specificity may beconserved by combining clones which possess the same H-CDR-3 regions.Further details are given in illustrative Example 13.

Preferred cross-cloned antibody molecules of the invention are selectedfrom the group consisting of MS-R #3.3H1×3.4L9, MS-R #3.4H1×3.4L9, MS-R#3.4H3×3.4L7, MS-R #3.4H3×3.4L9, MS-R #3.4H7×3.4L9, MS-R #3.4H7×3.4L7,MS-R #3.6H5×3.6L1, MS-R #3.6H5×3.6L2, MS-R #3.6.H8×3.6.L2, MS-R#7.2H2×7.2L1, MS-R #7.4H2×7.2L1, MS-R #7.4H2×7.12L2, MS-R#7.9H2×7.2L1(L1), MS-R #7.9H2×7.12L1, MS-R #7.9H2×7.12L2, MS-R#7.9H2×7.12L2(L1+2), MS-R #7.9H4×7.12.L2, MS-R #7.11H1×7.2L1, MS-R#7.11H1×7.11L1, MS-R #7.11H2×7.2L1(L1), MS-R #7.11H2×7.9L1 (L1), MS-R#7.11H2×7.12L1 or MS-R #8.1H1×8.2L1.

The generation of cross-cloned antibodies is also illustrated in theappended examples. The above mentioned preferred cross-clonedantibodies/antibody molecules are optimized/matured antibody moleculesderived from parental antibodies disclosed herein, in particular fromMSR-3 and MSR-7. in addition, further characterizing CDR-sequences andV-regions of the cross-cloned antibody molecules/antibodies are given inappended SEQ ID NOs: 32, 33, 46 and 47 (MSR 3.6H5×3.6.L2; V_(H)-,V_(L)-region); 34, 35, 48 and 49 (MSR 3.6H8×3.6.L2; V_(H)-,V_(L)-regions); 36, 37, 50 and 51 (MSR 7.4H2×7.2.L1; V_(H)-,V_(L)-regions); 38, 39, 52 and 53 (MSR 7.9H2×7.12.L2; V_(H)-,V_(L)-regions); 40, 41, 54 and 55 (MSR #7.9H4×7.12.L2; V_(H)-,V_(L)-regions); 42, 43, 56 and 57 (MSR #7.11H1×7.11.L1; V_(H)-,V_(L)-regions); and 44, 45, 58 and 59 (MSR #7.11H1×7.2.L1; V_(H)-,V_(L)-regions). Corresponding CDR3 regions of these particular preferredcross-cloned antibody molecules are depicted in SEQ ID NOs: 60 to 87.For further MSR antibody molecules, V_(H)-, V_(L)-, CDR-regions can bededuced from appended Tables 8 or 10 and from the appended sequencelisting, in particular SEQ ID NOS: 32 to 95 for MS-R antibodies/antibodymolecules #3.6H5×3.6L2, #3.6H8×3.6L2, #7.4H2×7.2L1, #7.9H2×7.12L2,#7.9H4×7.12L2, #7.11H1×7.11L1, #7.11H1×7.2L1 and #7.9H7 or SEQ ID NOS:294 to 413 for MSR-R antibodies/antibody molecules MS-R #3.3H1×3.4L9,#3.4H1×3.4L9, #3.4H3×3.4L7, #3.4H3×3.4L9, #3.4H7×3.4L9, #3.4H7×3.4L7,#3.6H5×3.6L1, #7.2H2×7.2L1, #7.4H2×7.12L2, #7.9H2×7.2L1, #7.9H2×7.12L1,#7.11H2×7.2L1, #7.11H2×7.9L1, #7.11H2×7.12L1 or #8.1H1×8.2L1.Accordingly, besides V_(H)-regions defined above, preferred antibodymolecules of the invention may comprise V_(H)-regions as defined in anyone of SEQ ID NOs: 294 to 323. Similarly, SEQ ID NOs: 324 to 353 depictpreferred V_(L)-regions which, besides to V_(L)-regions defined abovewhich may be comprised in the inventive antibody molecules.Corresponding CDR-3 regions are defined above, as well as in additionalsequences shown in SEQ ID NOs: 354 to 413.

Inventive antibody molecules can easily be produced in sufficientquantities, inter alia, by recombinant methods known in the art, see,e.g. Bentley, Hybridoma 17 (1998), 559-567; Racher, Appl. Microbiol.Biotechnol. 40 (1994), 851-856; Samuelsson, Eur. J. Immunol. 26 (1996),3029-3034.

Theoretically, in soluble β-A4 (monomeric/oligomeric) both theN-terminal and the middle epitopes are accessible for antibodyinteraction and antibody molecules of the present invention may eitherbind to the N-terminal or middle epitope separately, but under theseconditions maximum affinity will not be obtained. However, it is morelikely that an optimal contact to the antibody paratope will be attainedby simultaneous binding to both epitopes, i.e. similar to theinteraction with aggregated β-A4. Thus, antibodies of the presentinvention are unique anti-Aβ antibodies in that they bind to aggregatedβ-A4 (via interaction with the N-terminal and middle epitope), and atthe same time are also able to stabilize and neutralize theconformational epitope in soluble β-A4. These antibodies are distinct toprior art antibodies.

Most preferred are antibody molecules of the invention which have anaffinity to Aβ or defined fragments thereof with a K_(D) value lowerthan 2000 nM, preferably lower than 100 nM, more preferably lower than10 nM, most preferably lower than 1 nM. The measurement of suchaffinity/affinities may be carried out by methods illustrated in theexamples and known in the art. Such methods comprise, but are notlimited to BIACORE™-assays (www.biacore.com; Malmquist (1999), Biochem.Soc. Trans 27, 335-340) and solid phase assays using labeled antibodiesor labeled Aβ.

Preferably, the antibody molecule of the invention is capable ofdecorating/reacting with/binding to amyloid plaques in in vitro(post-mortem) brain sections from patients suffering fromamyloid-related disorders, like Alzheimer's disease. Yet, it is alsopreferred that the inventive antibody/antibody molecules preventAβ-aggregation in vivo as well as in in vitro assays, as illustrated inthe appended examples. Similarly, the antibody molecules of the presentinvention are preferred to de-polymerize Aβ-aggregate in vivo and/or inin vitro assays shown in the examples. This capacity of the inventiveantibodies/antibody molecules is, inter alia, to be employed in medicalsettings, in particular in pharmaceutical compositions described hereinbelow.

The invention also provides for a nucleic acid molecule encoding aninventive antibody molecule as defined herein.

Said nucleic acid molecule may be a naturally nucleic acid molecule aswell as a recombinant nucleic acid molecule. The nucleic acid moleculeof the invention may, therefore, be of natural origin, synthetic orsemi-synthetic. It may comprise DNA, RNA as well as PNA and it may be ahybrid thereof.

It is evident to the person skilled in the art that regulatory sequencesmay be added to the nucleic acid molecule of the invention. For example,promoters, transcriptional enhancers and/or sequences which allow forinduced expression of the polynucleotide of the invention may beemployed. A suitable inducible system is for exampletetracycline-regulated gene expression as described, e.g., by Gossen andBujard (Proc. Natl. Acad. Sci. USA 89 (1992), 5547-5551) and Gossen etal. (Trends Biotech. 12 (1994), 58-62), or a dexamethasone-induciblegene expression system as described, e.g. by Crook (1989) EMBO J. 8,513-519.

Furthermore, it is envisaged for further purposes that nucleic acidmolecule may contain, for example, thioester bonds and/or nucleotideanalogues. Said modifications may be useful for the stabilization of thenucleic acid molecule against endo- and/or exonucleases in the cell.Said nucleic acid molecules may be transcribed by an appropriate vectorcontaining a chimeric gene which allows for the transcription of saidnucleic acid molecule in the cell. In this respect, it is also to beunderstood that the polynucleotide of the invention can be used for“gene targeting” or “gene therapeutic” approaches. In another embodimentsaid nucleic acid molecules are labeled. Methods for the detection ofnucleic acids are well known in the art, e.g., Southern and Northernblotting, PCR or primer extension. This embodiment may be useful forscreening methods for verifying successful introduction of the inventivenucleic acid molecules during gene therapy approaches.

The nucleic acid molecule(s) of the invention may be a recombinantlyproduced chimeric nucleic acid molecule comprising any of theaforementioned nucleic acid molecules either alone or in combination.Preferably, the nucleic acid molecule of the invention is part of avector.

The present invention therefore also relates to a vector comprising thenucleic acid molecule of the present invention.

The vector of the present invention may be, e.g., a plasmid, cosmid,virus, bacteriophage or another vector used e.g. conventionally ingenetic engineering, and may comprise further genes such as marker geneswhich allow for the selection of said vector in a suitable host cell andunder suitable conditions.

Furthermore, the vector of the present invention may, in addition to thenucleic acid sequences of the invention, comprise expression controlelements, allowing proper expression of the coding regions in suitablehosts. Such control elements are known to the artisan and may include apromoter, a splice cassette, translation initiation codon, translationand insertion site for introducing an insert into the vector.Preferably, the nucleic acid molecule of the invention is operativelylinked to said expression control sequences allowing expression ineukaryotic or prokaryotic cells.

Control elements ensuring expression in eukaryotic and prokaryotic cellsare well known to those skilled in the art. As mentioned herein above,they usually comprise regulatory sequences ensuring initiation oftranscription and optionally poly-A signals ensuring termination oftranscription and stabilization of the transcript. Additional regulatoryelements may include transcriptional as well as translational enhancers,and/or naturally-associated or heterologous promoter regions. Possibleregulatory elements permitting expression in for example mammalian hostcells comprise the CMV-HSV thymidine kinase promoter, SV40, RSV-promoter(Rous Sarcoma Virus), human elongation factor 1α-promoter, theglucocorticoid-inducible MMTV-promoter (Moloney Mouse Tumor Virus),metallothionein- or tetracyclin-inducible promoters, or enhancers, likeCMV enhancer or SV40-enhancer. For expression in neural cells, it isenvisaged that neurofilament-, PGDF-, NSE-, PrP-, or thy-1-promoters canbe employed. Said promoters are known in the art and, inter alia,described in Charron (1995), J. Biol. Chem. 270, 25739-25745. For theexpression in prokaryotic cells, a multitude of promoters including, forexample, the tac-lac-promoter or the trp promoter, has been described.Besides elements which are responsible for the initiation oftranscription such regulatory elements may also comprise transcriptiontermination signals, such as SV40-poly-A site or the tk-poly-A site,downstream of the polynucleotide. In this context, suitable expressionvectors are known in the art such as Okayama-Berg cDNA expression vectorpcDV1 (Pharmacia), pRc/CMV, pcDNA1, pcDNA3 (In-vitrogene), pSPORT1(GIBCO BRL), pX (Pagano (1992) Science 255, 1144-1147), yeast two-hybridvectors, such as pEG202 and dpJG4-5 (Gyuris (1995) Cell 75, 791-803), orprokaryotic expression vectors, such as lambda gt11 or pGEX(Amersham-Pharmacia). Beside the nucleic acid molecules of the presentinvention, the vector may further comprise nucleic acid sequencesencoding for secretion signals. Such sequences are well known to theperson skilled in the art. Furthermore, depending on the expressionsystem used leader sequences capable of directing the peptides of theinvention to a cellular compartment may be added to the coding sequenceof the nucleic acid molecules of the invention and are well known in theart. The leader sequence(s) is (are) assembled in appropriate phase withtranslation, initiation and termination sequences, and preferably, aleader sequence capable of directing secretion of translated protein, ora protein thereof, into the periplasmic space or extracellular medium.Optionally, the heterologous sequence can encode a fusion proteinincluding an C- or N-terminal identification peptide imparting desiredcharacteristics, e.g., stabilization or simplified purification ofexpressed recombinant product. Once the vector has been incorporatedinto the appropriate host, the host is maintained under conditionssuitable for high level expression of the nucleotide sequences, and, asdesired, the collection and purification of the antibody molecules orfragments thereof of the invention may follow. The invention alsorelates, accordingly, to hosts/host cells which comprise a vector asdefined herein. Such hosts may be useful for in processes for obtainingantibodies/antibody molecules of the invention as well as inmedical/pharmaceutical settings. Said host cells may also comprisetransduced or transfected neuronal cells, like neuronal stem cells,preferably adult neuronal stem cells. Such host cells may be useful intransplantation therapies.

Furthermore, the vector of the present invention may also be anexpression, a gene transfer or gene targeting vector. Gene therapy,which is based on introducing therapeutic genes into cells by ex-vivo orin-vivo techniques is one of the most important applications of genetransfer. Transgenic mice expressing a neutralizing antibody directedagainst nerve growth factor have been generated using the“neuroantibody” technique; Capsoni, Proc. Natl. Acad. Sci. USA 97.(2000), 6826-6831 and Biocca, Embo J. 9 (1990), 101-108. Suitablevectors, methods or gene-delivering systems for in-vitro or in-vivo genetherapy are described in the literature and are known to the personskilled in the art; see, e.g., Giordano, Nature Medicine 2 (1996),534-539; Schaper, Circ. Res. 79 (1996), 911-919; Anderson, Science 256(1992), 808-813, Isner, Lancet 348 (1996), 370-374; Muhlhauser, Circ.Res. 77 (1995), 1077-1086; Onodua, Blood 91 (1998), 30-36; Verzeletti,Hum. Gene Ther. 9 (1998), 2243-2251; Verma, Nature 389 (1997), 239-242;Anderson, Nature 392 (Supp. 1998), 25-30; Wang, Gene Therapy 4 (1997),393-400; Wang, Nature Medicine 2 (1996), 714-716; WO 94/29469; WO97/00957; U.S. Pat. No. 5,580,859; U.S. Pat. No. 5,589,466; U.S. Pat.No. 4,394,448 or Schaper, Current Opinion in Biotechnology 7 (1996),635-640, and references cited therein. In particular, said vectorsand/or gene delivery systems are also described in gene therapyapproaches in neurological tissue/cells (see, inter alia Blömer, J.Virology 71 (1997) 6641-6649) or in the hypothalamus (see, inter alia,Geddes, Front Neuroendocrinol. (1999), 296-316 or Geddes, Nat. Med. 3(1997), 1402-1404). Further suitable gene therapy constructs for use inneurological cells/tissues are known in the art, for example in Meier(1999), J. Neuropathol. Exp. Neurol. 58, 1099-1110. The nucleic acidmolecules and vectors of the invention may be designed for directintroduction or for introduction via liposomes, viral vectors (e.g.adenoviral, retroviral), electroporation, ballistic (e.g. gene gun) orother delivery systems into the cell. Additionally, a baculoviral systemcan be used as eukaryotic expression system for the nucleic acidmolecules of the invention. The introduction and gene therapeuticapproach should, preferably, lead to the expression of a functionalantibody molecule of the invention, whereby said expressed antibodymolecule is particularly useful in the treatment, amelioration and/orprevention of neurological disorders related to abnormal amyloidsynthesis, assembly and/or aggregation, like, Alzheimer's disease andthe like.

Accordingly, the nucleic acid molecule of the present invention and/orthe above described vectors/hosts of the present invention may beparticularly useful as pharmaceutical compositions. Said pharmaceuticalcompositions may be employed in gene therapy approaches. In thiscontext, it is envisaged that the nucleic acid molecules and/or vectorsof the present invention may be employed to modulate, alter and/ormodify the (cellular) expression and/or concentration of the antibodymolecules of the invention or of (a) fragment(s) thereof.

For gene therapy applications, nucleic acids encoding the peptide(s) ofthe invention or fragments thereof may be cloned into a gene deliveringsystem, such as a virus and the virus used for infection and conferringdisease ameliorating or curing effects in the infected cells ororganism.

The present invention also relates to a host cell transfected ortransformed with the vector of the invention or a non-human hostcarrying the vector of the present invention, i.e. to a host cell orhost which is genetically modified with a nucleic acid moleculeaccording to the invention or with a vector comprising such a nucleicacid molecule. The term “genetically modified” means that the host cellor host comprises in addition to its natural genome a nucleic acidmolecule or vector according to the invention which was introduced intothe cell or host or into one of its predecessors/parents. The nucleicacid molecule or vector may be present in the genetically modified hostcell or host either as an independent molecule outside the genome,preferably as a molecule which is capable of replication, or it may bestably integrated into the genome of the host cell or host.

The host cell of the present invention may be any prokaryotic oreukaryotic cell. Suitable prokaryotic cells are those generally used forcloning like E. coli or Bacillus subtilis. Furthermore, eukaryotic cellscomprise, for example, fungal or animal cells. Examples for suitablefungal cells are yeast cells, preferably those of the genusSaccharomyces and most preferably those of the species Saccharomycescerevisiae. Suitable animal cells are, for instance, insect cells,vertebrate cells, preferably mammalian cells, such as e.g. HEK293, NSO,CHO, MDCK, U2-OSHela, NIH3T3, MOLT-4, Jurkat, PC-12, PC-3, IMR, NT2N,Sk-n-sh, CaSki, C33A. These host cells, e.g. CHO-cells, may providepost-translational modifications to the antibody molecules of theinvention, including leader peptide removal, folding and assembly of H(heavy) and L (light) chains, glycosylation of the molecule at correctsides and secretion of the functional molecule. Further suitable celllines known in the art are obtainable from cell line depositories, likethe American Type Culture Collection (ATCC). In accordance with thepresent invention, it is furthermore envisaged that primary cells/cellcultures may function as host cells. Said cells are in particularderived from insects (like insects of the species Drosophila or Blatta)or mammals (like human, swine, mouse or rat). Said host cells may alsocomprise cells from and/or derived from cell lines like neuroblastomacell lines. The above mentioned primary cells are well known in the artand comprise, inter alia, primary astrocytes, (mixed) spinal cultures orhippocampal cultures.

In a more preferred embodiment the host cell which is transformed withthe vector of the invention is a neuronal cell, a neuronal stem cell(e.g. an adult neuronal stem cell), a brain cell or a cell (line)derived therefrom. However, also a CHO-cell comprising the nucleic acidmolecule of the present invention may be particularly useful as host.Such cells may provide for correct secondary modifications on theexpressed molecules, i.e. the antibody molecules of the presentinvention. These modifications comprise, inter alia, glycosylations andphosphorylations.

Hosts may be non-human mammals, most preferably mice, rats, sheep,calves, dogs, monkeys or apes. Said mammals may be indispensable fordeveloping a cure, preferably a cure for neurological and/orneurodegenerative disorders mentioned herein. Furthermore, the hosts ofthe present invention may be particularly useful in producing theantibody molecules (or fragments thereof) of the invention. It isenvisaged that said antibody molecules (or fragments thereof) beisolated from said host. It is, inter alia, envisaged that the nucleicacid molecules and or vectors described herein are incorporated insequences for transgenic expression. The introduction of the inventivenucleic acid molecules as transgenes into non-human hosts and theirsubsequent expression may be employed for the production of theinventive antibodies. For example, the expression of such (a)transgene(s) in the milk of the transgenic animal provide for means ofobtaining the inventive antibody molecules in quantitative amounts; seeinter alia, U.S. Pat. No. 5,741,957, U.S. Pat. No. 5,304,489 or U.S.Pat. No. 5,849,992. Useful transgenes in this respect comprise thenucleic acid molecules of the invention, for example, coding sequencesfor the light and heavy chains of the antibody molecules describedherein, operatively linked to promotor and/or enhancer structures from amammary gland specific gene, like casein or beta-lactoglobulin.

The invention also provides for a method for the preparation of anantibody molecule of the invention comprising culturing the host celldescribed herein above under conditions that allow synthesis of saidantibody molecule and recovering said antibody molecule from saidculture.

The invention also relates to a composition comprising an antibodymolecule of the invention or produced by the method described hereinabove, a nucleic acid molecule encoding the antibody molecule of theinvention, a vector comprising said nucleic acid molecule or a host-cellas defined herein above and optionally, further molecules, either aloneor in combination, like e.g. molecules which are capable of interferingwith the formation of amyloid plaques or which are capable ofdepolymerizing already formed amyloid-plaques. The term “composition” asemployed herein comprises at least one compound of the invention.Preferably, such a composition is a pharmaceutical or a diagnosticcomposition.

The composition may be in solid or liquid form and may be, inter alia,in a form of (a) powder(s), (a) tablet(s), (a) solution(s) or (an)aerosol(s). Said composition may comprise on or more antibodies/antibodymolecules of the invention or nucleic acid molecules, vector or hosts ofthe invention. It is also envisaged that said composition comprises atleast two, preferably three, more preferably four, most preferably fiveantibody molecules of the invention or nucleic acid molecule(s) encodingsaid antibody molecule(s). Said composition may also comprise optimized,inventive antibodies/antibody molecules obtainable by the methodsdescribed herein below and in the appended examples.

It is preferred that said pharmaceutical composition, optionallycomprises a pharmaceutically acceptable carrier and/or diluent. Theherein disclosed pharmaceutical composition may be particularly usefulfor the treatment of neurological and/or neurodegenerative disorders.Said disorders comprise, but are not limited to Alzheimer's disease,amyothrophic lateral sclerosis (ALS), hereditary cerebral hemorrhagewith amyloidosis Dutch type, Down's syndrome, HIV-dementia, Parkinson'sdisease and neuronal disorders related to aging. The pharmaceuticalcomposition of the invention is, inter alia, envisaged as potentinhibitors of amyloid plaque formation or as a potent stimulator for thede-polymerization of amyloid plaques. Therefore, the present inventionprovides for pharmaceutical compositions comprising the compounds of theinvention to be used for the treatment of diseases/disorders associatedwith pathological APP proteolysis and/or amyloid plaque formation.

Examples of suitable pharmaceutical carriers, excipients and/or diluentsare well known in the art and include phosphate buffered salinesolutions, water, emulsions, such as oil/water emulsions, various typesof wetting agents, sterile solutions etc. Compositions comprising suchcarriers can be formulated by well known conventional methods. Thesepharmaceutical compositions can be administered to the subject at asuitable dose. Administration of the suitable compositions may beeffected by different ways, e.g., by intravenous, intraperitoneal,subcutaneous, intramuscular, topical, intradermal, intranasal orintrabronchial administration. It is particularly preferred that saidadministration is carried out by injection and/or delivery, e.g., to asite in a brain artery or directly into brain tissue. The compositionsof the invention may also be administered directly to the target site,e.g., by biolistic delivery to an external or internal target site, likethe brain. The dosage regimen will be determined by the attendingphysician and clinical factors. As is well known in the medical arts,dosages for any one patient depends upon many factors, including thepatient's size, body surface area, age, the particular compound to beadministered, sex, time and route of administration, general health, andother drugs being administered concurrently. Proteinaceouspharmaceutically active matter may be present in amounts between 1 ngand 10 mg/kg body weight per dose; however, doses below or above thisexemplary range are envisioned, especially considering theaforementioned factors. If the regimen is a continuous infusion, itshould also be in the range of 1 μg to 10 mg units per kilogram of bodyweight per minute.

Progress can be monitored by periodic assessment. The compositions ofthe invention may be administered locally or systemically. It is of notethat peripherally administered antibodies can enter the central nervoussystem, see, inter alia, Bard (2000), Nature Med. 6, 916-919.Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like. Furthermore, the pharmaceutical composition of theinvention may comprise further agents depending on the intended use ofthe pharmaceutical composition. Said agents may be drugs acting on thecentral nervous system, like, neuroprotective factors, cholinesteraseinhibitors, agonists of M1 muscarinic receptor, hormones, antioxidants,inhibitors of inflammation etc. It is particularly preferred that saidpharmaceutical composition comprises further agents like, e.g.neurotransmitters and/or substitution molecules for neurotransmitters,vitamin E, or alpha-lipoic acid.

The pharmaceutical compositions, as well as the methods of the inventionor the uses of the invention described infra can be used for thetreatment of all kinds of diseases hitherto unknown or being related toor dependent on pathological APP aggregation or pathological APPprocessing. They may be particularly useful for the treatment ofAlzheimer's disease and other diseases where extracellular deposits ofamyloid-β, appear to play a role. They may be desirably employed inhumans, although animal treatment is also encompassed by the methods,uses and compositions described herein.

In a preferred embodiment of the invention, the composition of thepresent invention as disclosed herein above is a diagnostic compositionfurther comprising, optionally, suitable means for detection. Thediagnostic composition comprises at least one of the aforementionedcompounds of the invention.

Said diagnostic composition may comprise the compounds of the invention,in particular and preferably the antibody molecules of the presentinvention, in soluble form/liquid phase but it is also envisaged thatsaid compounds are bound to/attached to and/or linked to a solidsupport.

Solid supports may be used in combination with the diagnosticcomposition as defined herein or the compounds of the present inventionmay be directly bound to said solid supports. Such supports are wellknown in the art and comprise, inter alia, commercially available columnmaterials, polystyrene beads, latex beads, magnetic beads, colloid metalparticles, glass and/or silicon chips and surfaces, nitrocellulosestrips, membranes, sheets, duracytes, wells and walls of reaction trays,plastic tubes etc. The compound(s) of the invention, in particular theantibodies of the present invention, may be bound to many differentcarriers. Examples of well-known carriers include glass, polystyrene,polyvinyl chloride, polypropylene, polyethylene, polycarbonate, dextran,nylon, amyloses, natural and modified celluloses, polyacrylamides,agaroses, and magnetite. The nature of the carrier can be either solubleor insoluble for the purposes of the invention. Appropriate labels andmethods for labeling have been identified above and are furthermorementioned herein below. Suitable methods for fixing/immobilizing saidcompound(s) of the invention are well known and include, but are notlimited to ionic, hydrophobic, covalent interactions and the like.

It is particularly preferred that the diagnostic composition of theinvention is employed for the detection and/or quantification of APPand/or APP-processing products, like amyloid-β or for the detectionand/or quantification of pathological and/or (genetically) modifiedAPP-cleavage sides.

As illustrated in the appended examples, the compounds of the presentinvention, in particular the inventive antibody molecules areparticularly useful as diagnostic reagents in the detection of genuinehuman amyloid plaques in brain sections of Alzheimer's Disease patientsby indirect immunofluorescence.

It is preferred that said compounds of the present invention to beemployed in a diagnostic composition are detectably labeled. A varietyof techniques are available for labeling biomolecules, are well known tothe person skilled in the art and are considered to be within the scopeof the present invention. Such techniques are, e.g., described inTijssen, “Practice and theory of enzyme immuno assays”, Burden, R H andvon Knippenburg (Eds), Volume 15 (1985), “Basic methods in molecularbiology”; Davis L G, Dibmer M D; Battey Elsevier (1990), Mayer et al.,(Eds) “Immunochemical methods in cell and molecular biology” AcademicPress, London (1987), or in the series “Methods in Enzymology”, AcademicPress, Inc.

There are many different labels and methods of labeling known to thoseof ordinary skill in the art. Examples of the types of labels which canbe used in the present invention include enzymes, radioisotopes,colloidal metals, fluorescent compounds, chemiluminescent compounds, andbioluminescent compounds.

Commonly used labels comprise, inter alia, fluorochromes (likefluorescein, rhodamine, Texas Red, etc.), enzymes (like horse radishperoxidase, β-galactosidase, alkaline phosphatase), radioactive isotopes(like ³²P or ¹²⁵I), biotin, digoxygenin, colloidal metals, chemi- orbioluminescent compounds (like dioxetanes, luminol or acridiniums).Labeling procedures, like covalent coupling of enzymes or biotinylgroups, iodinations, phosphorylations, biotinylations, etc. are wellknown in the art.

Detection methods comprise, but are not limited to, autoradiography,fluorescence microscopy, direct and indirect enzymatic reactions, etc.Commonly used detection assays comprise radioisotopic ornon-radioisotopic methods. These comprise, inter alia, Westernblotting,overlay-assays, RIA (Radioimmuno Assay) and IRMA (ImmuneRadioimmunometric Assay), EIA (Enzyme Immuno Assay), ELISA (EnzymeLinked Immuno Sorbent Assay), FIA (Fluorescent Immuno Assay), and CLIA(Chemioluminescent Immune Assay).

Furthermore, the present invention provides for the use of an antibodymolecule of invention, or an antibody molecule produced by the method ofthe invention, of a nucleic acid molecule, vector of or a host of theinvention for the preparation of a pharmaceutical or a diagnosticcomposition for the prevention, treatment and/or diagnosis of a diseaseassociated with amyloidogenesis and/or amyloid-plaque formation. It isfurther preferred that the compounds described herein, in particular theantibody molecules of the invention, be employed in the preventionand/or treatment of neuropathologies associated with modified orabnormal APP-processing and/or amyloidogenesis. The antibody molecules,e.g in format of (engineered) immunoglobulins, like antibodies in a IgGframework, in particular in an IgG1-framework, or in the format ofchimeric antibodies, bispecific antibodies, single chain Fvs (scFvs) orbispecific scFvs and the like are to employed in the preparation of thepharmaceutical compositions provided herein. Yet, the antibody moleculesare also useful in diagnostic settings as documented in the appendedexamples, since the antibody molecules of the invention specificallyinteract with/detect Aβ4 and/or amyloid deposits/plaques.

Therefore an inventive use of the compounds of the present invention isthe use for the preparation of a pharmaceutical composition for aneurological disorder which calls for amelioration, for example bydisintegration of β-amyloid plaques, by amyloid (plaque) clearance or bypassive immunization against β-amyloid plaque formation. As illustratedin the appended examples, the inventive antibody molecules areparticularly useful in preventing Aβ aggregation and inde-polymerization of already formed amyloid aggregates. Accordingly, theinventive antibodies are to be employed in the reduction of pathologicalamyloid deposits/plaques, in the clearance of amyloid plaques/plaqueprecursors as well as in neuronal protection. It is in particularenvisaged that the antibody molecules of the invention be employed inthe in vivo prevention of amyloid plaques as well as in in vivoclearance of pre-existing amyloid plaques/deposits. Furthermore, theantibody molecules of the invention may be employed in passiveimmunization approaches against Aβ4. Clearance of Aβ4/Aβ4 deposits may,inter alia, be achieved by the medical use of antibodies of the presentinvention which comprise an Fc-part. Said Fc-part of an antibody may beparticularly useful in Fc-receptor mediated immune responses, e.g. theattraction of macrophages (phagocytic cells and/or microglia) and/orhelper cells. For the mediation of Fc-part-related immunoresponses, theantibody molecule of the invention is preferably in an (human)IgG1-framework. As discussed herein, the preferred subject to be treatedwith the inventive antibody molecules, the nucleic acid moleculesencoding the same or parts thereof, the vectors of the invention or thehost cells of this invention is a human subject. Other frameworks, likeIgG2a- or IgG2b-frameworks for the inventive antibody molecules are alsoenvisaged. Immunoglobulin frameworks in IgG2a and IgG2b format areparticular envisaged in mouse settings, for example in scientific usesof the inventive antibody molecules, e.g. in tests on transgenic miceexpressing (human) wildtype or mutated APP, APP-fragments and/or Aβ4.

The above recited diseases associated with amyloidogenesis and/oramyloid-plaque formation comprise, but are not limited to dementia,Alzheimer's disease, motor neuropathy, Parkinson's disease, ALS(amyotrophic lateral sclerosis), scrapie, HIV-related dementia as wellas Creutzfeld-Jakob disease, hereditary cerebral hemorrhage, withamyloidis Dutch type, Down's syndrome and neuronal disorders related toaging. The antibody molecules of the invention and the compositionsprovided herein may also be useful in the amelioration and or preventionof inflammatory processes relating to amyloidogenesis and/or amyloidplaque formation.

Accordingly, the present invention also provides for a method fortreating, preventing and/or delaying neurological and/orneurodegenerative disorders comprising the step of administering to asubject suffering from said neurological and/or neurodegenerativedisorder and/or to a subject susceptible to said neurological and/orneurodegenerative disorder an effective amount of a antibody molecule ofthe invention, a nucleic acid molecule of invention and/or a compositionas defined herein above.

In yet another embodiment, the present invention provides for a kitcomprising at least one antibody molecule, at least one nucleic acidmolecule, at least one vector or at least one host cell of theinvention. Advantageously, the kit of the present invention furthercomprises, optionally (a) buffer(s), storage solutions and/or remainingreagents or materials required for the conduct of medical, scientific ordiagnostic assays and purposes. Furthermore, parts of the kit of theinvention can be packaged individually in vials or bottles or incombination in containers or multicontainer units.

The kit of the present invention may be advantageously used, inter alia,for carrying out the method of the invention and could be employed in avariety of applications referred herein, e.g., as diagnostic kits, asresearch tools or medical tools. Additionally, the kit of the inventionmay contain means for detection suitable for scientific, medical and/ordiagnostic purposes. The manufacture of the kits follows preferablystandard procedures which are known to the person skilled in the art.

The invention also provides for a method for the optimization of anantibody molecule as defined herein above comprising the steps of

(a) constructing a library of diversified Fab antibody fragments derivedfrom an antibody comprising at least one CDR3 of an V_(H)-region asencoded by a nucleic acid molecule as shown in SEQ ID NOs: 21, 23 or 25or at least one CDR3 amino acid sequence of an V_(H)-region as shown inSEQ ID NOs: 22, 24 or 26;(b) testing the resulting Fab optimization library by panning againstAβ/Aβ4;(c) identifying optimized clones; and(d) expressing of selected, optimized clones.

Optimization of the antibodies/antibody molecules of the invention isalso documented in the appended examples and may comprise the selectionfor, e.g. higher affinity for one or both regions/epitopes of β-A4 asdefined herein or selection for improved expression and the like. In oneembodiment, said selection for to higher affinity for one or bothregions/epitopes of β-A4 comprises the selection for high affinity to(a) an amino acid stretch comprising amino acids 2 to 10 (or (a) part(s)thereof) of β-A4 and/or (b) an amino acid stretch comprising amino acids12 to 25 (or (a) part(s) thereof) of β-A4 (SEQ ID NO. 27).

The person skilled in the art can readily carry out the inventive methodemploying the teachings of the present invention. Optimization protocolsfor antibodies are known in the art. These optimization protocolscomprise, inter alia, CDR walking mutagenesis as disclosed andillustrated herein and described in Yang (1995), J. Mol. Biol. 25,392-403; Schier (1996), J. Mol. Biol. 263, 551-567; Barbas (1996),Trends. Biotech 14, 230-34 or Wu (1998), PNAS 95, 6037-6042; Schier(1996), Human Antibodies Hybridomas 7, 97; Moore (1997), J. Mol. Biol.272, 336.

“Panning”-techniques are also known in the art, see, e.g. Kay (1993),Gene 128, 59-65. Furthermore, publications like Borrebaeck (1995),“Antibody Engineering”, Oxford University, 229-266; McCafferty (1996),“Antibody Engineering”, Oxford University Press; Kay (1996), ALaboratory Manual, Academic Press provide for optimization protocolswhich may be modified in accordance with this invention.

The optimization method may further comprise a step (ca), whereby theoptimized clones are further optimized by cassette mutagenesis, asillustrated in the appended examples.

The method for the optimization of an antibody molecule described hereinis further illustrated in the appended examples as affinity maturationof parental antibodies/antibody molecules capable of specificallyrecognizing two regions of the beta-A4 peptide/Abeta4/Aβ/Aβ4/βA4.

Preferably, said Aβ/Aβ4 (also designated as βA4 in context of thisinvention) in step (b) of the method described herein above isaggregated Aβ/Aβ4. Said panning may be carried out (as described in theappended examples) with increased stringency of binding. Stringency maybe increased, inter alia, by reducing the Aβ/Aβ4 concentration or byelevating the (assay) temperature. The testing of the optimized libraryby panning is known to the skilled artisan and described in Kay (1993),loc. cit. Preferably, the identification in step (c) is carried out byranking according to the lowest K_(D)-values.

Most preferably said identification in step (c) is carried out bykoff-ranking. Koff-ranking is known to the skilled artisan and describedin Schier (1996), loc. cit.; Schier (1996), J. Mol. Biol. 255, 28-43 orDuenas (1996), Mol. Immunol. 33, 279-286. Furthermore, koff-ranking isillustrated in the appended examples. The off-rate constant may bemeasured as described in the appended examples.

As mentioned herein above, the identified clones may, for furtherevaluation, be expressed. The expression may be carried out by knownmethods, inter alia, illustrated in the appended examples. Theexpression may, inter alia, lead to expressed Fab-fragments, scFvs,bispecific immunoglobulins, bispecific antibody molecules, Fab- and/orFv fusion proteins, or full antibodies, like IgGs, in particular IgG1.

Optimized antibodies, in particular optimized Fabs or optimized IgGs,preferably IgG1s, may be tested by methods as illustrated in theappended examples. Such methods comprise, but are not limited to, thetesting of binding affinities, the determination of K_(D) values,pepspot analysis, ELISA-assays, RIA-assays, CLIA-assays, (immuno-)histological studies (for example staining of amyloid plaques),de-polymerization assays or antibody-dependent β-A4 phagocytoses.

In a further embodiment of the present invention, a method is providedwherein optimized antibodies are generated by cross-cloning. This methodis also illustrated in the appended examples and comprises the step ofcombining independently optimized CDR-regions, for example, by combiningindependently optimized H-CDR2 and L-CDR2 from matured clones withH-CDR3, preferably the same H-CDR3.

In a preferred embodiment, the invention relates to a method for thepreparation of a pharmaceutical composition comprising the steps of

(a) optimization of an antibody according to the method described hereinand illustrated in the appended examples; and(b) formulating the optimized antibody/antibody molecule with anphysiologically acceptable carrier, as described herein above.

Accordingly, the invention also provides for a pharmaceuticalcomposition prepared by the method disclosed herein and comprisingfurther optimized antibody molecules capable of specifically recognizingtwo regions of the beta-A4 peptide/Abeta4/Aβ/A4β/βA4, as describedherein above.

Exemplified Sequences as Recited Herein:

SEQ ID NO: 1 AEFRHDSGY First region of β-A4 peptide, “N-terminalregion/epitope” SEQ ID NO: 2 VHHQKLVFFAEDVG Second region of β-A4peptide, “Central/middle region/epitope” VH-region of MS-Roche#3(nucleic acid sequence) SEQ ID NO: 3CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCGGCAGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTCTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGC (SEQ ID NO: 3) VH-region of MS-Roche#3 (aminoacid sequence) SEQ ID NO: 4QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTHYARYYRYFDVWGQGTL VTVSS (SEQID NO: 4) VH-region of MS-Roche#7 (nucleic acid sequence) SEQ ID NO: 5CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCGGCAGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGC (SEQ ID NO: 5) VH-region ofMS-Roche#7 (amino acid sequence) SEQ ID NO: 6QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWG QGTLVTVSS(SEQ ID NO: 6) VH-region of MS-Roche#8 (nucleic acid sequence) SEQ IDNO: 7 CAGGTGCAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCGATTAGCGGTAGCGGCGGCAGCACCTATTATGCGGATAGCGTGAAAGGCCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTCTTCTTTCTCGTGGTTATAATGGTTATTATCATAAGTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCAGC (SEQ ID NO: 7) VH-region of MS-Roche#8(amino acid sequence) SEQ ID NO: 8QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLLSRGYNGYYHKFDVWGQG TLVTVSS (SEQID NO: 8) VL-region of MS-Roche#3 (nucleic acid sequence) SEQ ID NO: 9GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGGTTTATAATCCTCCTGTTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 9) VL-regionof MS-Roche #3 (amino acid sequence) SEQ ID NO: 10DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQVYNPPVTFGQGTKVEIKRT (SEQ ID NO: 10)VL-region of MS-Roche#7 (nucleic acid sequence) SEQ ID NO: 11GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCTTTCAGCTTTATTCTGATCCTTTTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO. 11) VL-regionof MS-Roche#7 (amino acid sequence) SEQ ID NO: 12DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCFQLYSDPFTFGQGTKVEIKRT (SEQ ID NO: 12)VL-region of MS-Roche#8 (nucleic acid sequence) SEQ ID NO: 13GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGAGCGTGAGCAGCAGCTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGCTTTCTTCTTTTCCTCCTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 13) VL-regionof MS-Roche#8 (amino acid sequence) SEQ ID NO: 14DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQQLSSFPPTFGQGTKVEIKRT (SEQ ID NO: 14) CDR3of V_(L)-region of MSR-3 (nucleic acid sequence) SEQ ID NO: 15|CAGCAGGTTTATAATCCTCCTGTT| (SEQ ID NO: 15) CDR3 of V_(L)-region of MSR-3(amino acid sequence) SEQ ID NO: 16 QQVYNPPV (SEQ ID NO: 16) CDR3 ofV_(L)-region of MSR-7 (nucleic acid sequence) SEQ ID NO: 17|TTTCAGCTTTATTCTGATCCTTTT| (SEQ ID NO: 17) CDR3 of V_(L)-region of MSR-7(amino acid sequence) SEQ ID NO: 18 FQLYSDPF (SEQ ID NO. 18) CDR3 ofV_(L)-region of MSR-8 (nucleic acid sequence) SEQ ID NO: 19 CAG CAG CTTTCT TCT TTT CCT CCT (SEQ ID NO. 19) CDR3 of V_(L)-region of MSR-8 (aminoacid sequence) SEQ ID NO: 20 QQLSSFPP (SEQ ID NO: 20) CDR ofV_(H)-region of MSR-3 (nucleic acid sequence) SEQ ID NO: 21CTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTT (SEQ ID NO: 21) CDR ofV_(H)-region of MSR-3 (amino acid sequence) SEQ ID NO: 22 LTHYARYYRYFDV(SEQ ID NO: 22) CDR of V_(H)-region of MSR-7 (nucleic acid sequence) SEQID NO: 23 GGT AAG GGT AAT ACT CAT AAG CCT TAT GGT TAT GTT CGT TAT TTTGAT GTT (SEQ ID NO: 23) CDR of V_(H)-region of MSR-7 (amino acidsequence) SEQ ID NO: 24 GKGNTHKPYGYVRYFDV (SEQ ID NO: 24) CDR ofV_(H)-region of MSR-8 (nucleic acid sequence) SEQ ID NO: 25 CTT CTT TCTCGT GGT TAT AAT GGT TAT TAT CAT AAG TTT GAT GTT (SEQ ID NO. 25) CDR ofV_(H)-region of MSR-8 (amino acid sequence) SEQ ID NO: 26LLSRGYNGYYHKFDV (SEQ ID NO: 26) Aβ4 (amino acids 1 to 42) SEQ ID NO: 27DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVVIA (SEQ ID NO: 27) primer SEQ IDNO: 28 5′-GTGGTGGTTCCGATATC-3′ (SEQ ID NO: 28) primer SEQ ID NO: 295′-AGCGTCACACTCGGTGCGGCTTTCGGCTGGCCAAGAACGGTTA-3′ (SEQ ID NO: 29) primerSEQ ID NO: 30 5′-CAGGAAACAGCTATGAC-3′ (SEQ ID NO: 30) primer SEQ ID NO:31 5′-TACCGTTGCTCTTCACCCC-3′ (SEQ ID NO: 31) VH of MS-Roche#3.6H5 x3.6L2; DNA; artificial sequence SEQ ID NO: 32CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTTCTGAGTCTGGTAAGACTAAGTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTCTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 32) prot VH region of MS-Roche#3.6H5 x3.6L2; protein/1; artificial sequence SEQ ID NO. 33:QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISESGKTKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTHYARYYRYFDVWGQGTLVTVS S (SEQ IDNO: 33) VH region of MS-Roche#3.6H8 x 3.6L2; DNA; artificial sequenceSEQ ID NO: 34 CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTTCTGAGTATTCTAAGTTTAAGTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTCTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 34) prot VH region of MS-Roche#3.6H8 x 3.6L2;protein/1; artificial sequence SEQ ID NO: 35QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISEYSKFKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARLTHYARYYRYFDVWGQGTLVTVS S (SEQ IDNO: 35) VH region of MS-Roche#7.4H2 x 7.2L1; DNA; artificial sequenceSEQ ID NO: 36 CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATTATAATGGTGCTCGTATTTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 36) prot VH region ofMS-Roche#7.4H2 x 7.2L1; protein/1; artificial sequence SEQ ID NO: 37QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINYNGARIYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQGT LVTVSS (SEQID NO: 37) VH region of MS-Roche#7.9H2 x 7.12 L2; DNA; artificialsequence SEQ ID NO: 38CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATGCTGATGGTAATCGTAAGTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 38) prot VH region ofMS-Roche#7.9H2 x 7.12 L2; protein/1; artificial sequence SEQ ID NO: 39QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINADGNRKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS (SEQID NO: 39) VH region of MS-Roche#7.9H4 x 7.12L2; DNA; artificialsequence SEQ ID NO: 40CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGCTATTAATGCTGTTGGTATGAAGAAGTTTTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 40) prot VH region ofMS-Roche#7.9H4 x 7.12L2; protein/1; artificial sequence SEQ ID NO: 41QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINAVGMKKFYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS (SEQID NO: 41) VH region of MS-Roche#7.11H1 x 7.11L1; DNA; artificialsequence SEQ ID NO: 42CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATTAATGCTGCTGGTTTTCGTACTTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 42) prot VH region ofMS-Roche#7.11H1 x 7.11L1; protein/1; artificial sequence SEQ ID NO. 43QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINAAGFRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS (SEQID NO: 43) VH region of MS-Roche#7.11H1 x 7.2L1; DNA; artificialsequence SEQ ID NO: 44CAATTGGTGGAAAGCGGCGGCGGCCTGGTGCAACCGGGCGGCAGCCTGCGTCTGAGCTGCGCGGCCTCCGGATTTACCTTTAGCAGCTATGCGATGAGCTGGGTGCGCCAAGCCCCTGGGAAGGGTCTCGAGTGGGTGAGCGGTATTAATGCTGCTGGTTTTCGTACTTATTATGCTGATTCTGTTAAGGGTCGTTTTACCATTTCACGTGATAATTCGAAAAACACCCTGTATCTGCAAATGAACAGCCTGCGTGCGGAAGATACGGCCGTGTATTATTGCGCGCGTGGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTTTGGGGCCAAGGCACCCTGGTGACGGTTAGCTCA (SEQ ID NO: 44) prot VH region ofMS-Roche#7.11H1 x 7.2L1; protein/1; artificial sequence SEQ ID NO: 45QLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSGINAAGFRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWGQG TLVTVSS (SEQID NO: 45) VL region of MS-Roche#3.6H5 x 3.6L2; DNA; artificial sequenceSEQ ID NO: 46 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGTTTCTTTCTCGTTATTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGACTTATAATTATCCTCCTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 46) prot VLregion of MS-Roche#3.6H5 x 3.6L2; protein/1; artificial sequence SEQ IDNO: 47 DIVLTQSPATLSLSPGERATLSCRASQFLSRYYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQTYNYPPTFGQGTKVEIKRT (SEQ ID NO: 47) VLregion of MS-Roche#3.6H8 x 3.6L2; DNA; artificial sequence SEQ ID NO: 48GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGTTTCTTTCTCGTTATTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCAGCAGACTTATAATTATCCTCCTACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 48) prot VLregion of MS-Roche#3.6H8 x 3.6L2; protein/1; artificial sequence SEQ IDNO: 49 DIVLTQSPATLSLSPGERATLSCRASQFLSRYYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQTYNYPPTFGQGTKVEIKRT (SEQ ID NO: 49) VLregion of MS-Roche#7.4H2 x 7.2L1; DNA; artificial sequence SEQ ID NO: 50GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGTATGTTGATCGTACTTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGATTTATTCTTTTCCTCATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 50) prot VLregion of MS-Roche#7.4H2 x 7.2L1; protein/1; artificial sequence SEQ IDNO: 51 DIVLTQSPATLSLSPGERATLSCRASQYVDRTYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQQIYSFPHTFGQGTKVEIKRT (SEQ ID NO: 51) VLregion of MS-Roche#7.9H2 x 7.12 L2; DNA; artificial sequence SEQ ID NO:52 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGCGTTTTTTTTATAAGTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTCTGGTTCTTCTAACCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCTTCAGCTTTATAATATTCCTAATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 52) prot VLregion of MS-Roche#7.9H2 x 7.12 L2; protein/1; artificial sequence SEQID NO: 53 DIVLTQSPATLSLSPGERATLSCRASQRFFYKYLAWYQQKPGQAPRLLISGSSNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQLYNIPNTFGQGTKVEIKRT (SEQ ID NO: 53) VLregion of MS-Roche#7.9H4 x 7.12L2; DNA; artificial sequence SEQ ID NO:54 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGCGTTTTTTTTATAAGTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTCTGGTTCTTCTAACCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGGTTTATTATTGCCTTCAGCTTTATAATATTCCTAATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 54) prot VLregion of MS-Roche#7.9H4 x 7.12L2; protein/1; artificial sequence SEQ IDNO: 55 DIVLTQSPATLSLSPGERATLSCRASQRFFYKYLAWYQQKPGQAPRLLISGSSNRATGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCLQLYNIPNTFGQGTKVEIKRT (SEQ ID NO: 55) VLregion of MS-Roche#7.11H1 x 7.11L1; DNA; artificial sequence SEQ ID NO:56 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGCGTATTCTTCGTATTTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGGTTTATTCTCCTCCTCATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 56) prot VLregion of MS-Roche#7.11H1 x 7.11L1; protein/1; artificial sequence SEQID NO: 57 DIVLTQSPATLSLSPGERATLSCRASQRILRIYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQQVYSPPHTFGQGTKVEIKRT (SEQ ID NO: 57) VLregion of MS-Roche#7.11H1 x 7.2L1; DNA; artificial sequence SEQ ID NO:58 GATATCGTGCTGACCCAGAGCCCGGCGACCCTGAGCCTGTCTCCGGGCGAACGTGCGACCCTGAGCTGCAGAGCGAGCCAGTATGTTGATCGTACTTATCTGGCGTGGTACCAGCAGAAACCAGGTCAAGCACCGCGTCTATTAATTTATGGCGCGAGCAGCCGTGCAACTGGGGTCCCGGCGCGTTTTAGCGGCTCTGGATCCGGCACGGATTTTACCCTGACCATTAGCAGCCTGGAACCTGAAGACTTTGCGACTTATTATTGCCAGCAGATTTATTCTTTTCCTCATACCTTTGGCCAGGGTACGAAAGTTGAAATTAAACGTACG (SEQ ID NO: 58) prot VLregion of MS-Roche#7.11H1 x 7.2L1; protein/1; artificial sequence SEQ IDNO: 59 DIVLTQSPATLSLSPGERATLSCRASQYVDRTYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCQQIYSFPHTFGQGTKVEIKRT (SEQ ID NO: 59) HCDR3region of MS-Roche#3.6H5 x 3.6L2; DNA; artificial sequence SEQ ID NO: 60CTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTT (SEQ ID NO: 60) prot HCDR3region of MS-Roche#3.6H5 x 3.6L2; protein/1; artificial sequence SEQ IDNO: 61 LTHYARYYRYFDV (SEQ ID NO: 61) HCDR3 region of MS-Roche#3.6H8 x3.6L2; DNA; artificial sequence SEQ ID NO: 62CTTACTCATTATGCTCGTTATTATCGTTATTTTGATGTT (SEQ ID NO: 62) prot HCDR3region of MS-Roche#3.6H8 x 3.6L2; protein/1; artificial sequence SEQ IDNO: 63 LTHYARYYRYFDV (SEQ ID NO: 63) HCDR3 region of MS-Roche#7.4H2 x7.2L1; DNA; artificial sequence SEQ ID NO: 64GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT (SEQ ID NO: 64) protHCDR3 region of MS-Roche#7.4H2 x 7.2L1; protein/1; artificial sequenceSEQ ID NO: 65 GKGNTHKPYGYVRYFDV (SEQ ID NO: 65) HCDR3 region ofMS-Roche#7.9H2 x 7.12 L2; DNA; artificial sequence SEQ ID NO: 66GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT (SEQ ID NO: 66) protHCDR3 region of#MS-Roche 7.9H2 x 7.12 L2; protein/1; artificial sequenceSEQ ID NO: 67 GKGNTHKPYGYVRYFDV (SEQ ID NO: 67) HCDR3 region ofMS-Roche#7.9H4 x 7.12L2; DNA; artificial sequence SEQ ID NO: 68GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT (SEQ ID NO: 68) protHCDR3 region of MS-Roche#7.9H4 x 7.12L2; protein/1; artificial sequenceSEQ ID NO: 69 GKGNTHKPYGYVRYFDV (SEQ ID NO: 69) HCDR3 region ofMS-Roche#7.11H1 x 7.11L1; DNA; artificial sequence SEQ ID NO: 70GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT (SEQ ID NO: 70) protHCDR3 region of MS-Roche#7.11H1 x 7.11L1; protein/1; artificial sequenceSEQ ID NO: 71 GKGNTHKPYGYVRYFDV (SEQ ID NO: 71) HCDR3 region ofMS-Roche#7.11H1 x 7.2L1; DNA; artificial sequence SEQ ID NO: 72GGTAAGGGTAATACTCATAAGCCTTATGGTTATGTTCGTTATTTTGATGTT (SEQ ID NO: 72) protHCDR3 region of MS-Roche#7.11H1 x 7.2L1; protein/1; artificial sequenceSEQ ID NO: 73 GKGNTHKPYGYVRYFDV (SEQ ID NO: 73) LCDR3 region ofMS-Roche#3.6H5 x 3.6L2; DNA; artificial sequence SEQ ID NO: 74CAGCAGACTTATAATTATCCTCCT (SEQ ID NO: 74) prot LCDR3 region ofMS-Roche#3.6H5 x 3.6L2; protein/1; artificial sequence SEQ ID NO: 75QQTYNYPP (SEQ ID NO: 75) LCDR3 region of MS-Roche#3.6H8 x 3.6L2; DNA;artificial sequence SEQ ID NO: 76 CAGCAGACTTATAATTATCCTCCT (SEQ ID NO:76) prot LCDR3 region of MS-Roche#3.6H8 x 3.6L2; protein/1; artificialsequence SEQ ID NO: 77 QQTYNYPP (SEQ ID NO: 77) LCDR3 region ofMS-Roche#7.4H2 x 7.2L1; DNA; artificial sequence SEQ ID NO: 78CAGCAGATTTATTCTTTTCCTCAT (SEQ ID NO: 78) prot LCDR3 region ofMS-Roche#7.4H2 x 7.2L1; protein/1; artificial sequence SEQ ID NO: 79QQIYSFPH (SEQ ID NO: 79) LCDR3 region of MS-Roche#7.9H2 x 7.12 L2; DNA;artificial sequence SEQ ID NO: 80 CTTCAGCTTTATAATATTCCTAAT (SEQ ID NO:80) prot LCDR3 region of MS-Roche#7.9H2 x 7.12 L2; protein/1; artificialsequence SEQ ID NO: 81 LQLYNIPN (SEQ ID NO: 81) LCDR3 region ofMS-Roche#7.9H4 x 7.12L2; DNA; artificial sequence SEQ ID NO: 82CTTCAGCTTTATAATATTCCTAAT (SEQ ID NO: 82) prot LCDR3 region ofMS-Roche#7.9H4 x 7.12L2; protein/1; artificial sequence SEQ ID NO: 83LQLYNIPN (SEQ ID NO: 83) LCDR3 region of MS-Roche#7.11H1 x 7.11L1; DNA;artificial sequence SEQ ID NO: 84 CAGCAGGTTTATTCTCCTCCTCAT (SEQ ID NO:84) prot LCDR3 region of MS-Roche#7.11H1 x 7.11L1; protein/1; artificialsequence SEQ ID NO: 85 QQVYSPPH (SEQ ID NO: 85) LCDR3 region ofMS-Roche#7.11H1 x 7.2L1; DNA; artificial sequence SEQ ID NO: 86CAGCAGATTTATTCTTTTCCTCAT (SEQ ID NO: 86) prot LCDR3 region ofMS-Roche#7.11H1 x 7.2L1; protein/1; artificial sequence SEQ ID NO: 87QQIYSFPH (SEQ ID NO: 87) VH region of MS-Roche#7.9H7; DNA; artificialsequence SEQ ID NO: 88Caggtgcaattggtggaaagcggcggcggcctggtgcaaccgggcggcagcctgcgtctgagctgcgcggcctccggatttacctttagcagctatgcgatgagctgggtgcgccaagcccctgggaagggtctcgagtgggtgagcgctattaatgcttctggtactcgtacttattatgctgattctgttaagggtcgttttaccatttcacgtgataattcgaaaaacaccctgtatctgcaaatgaacagcctgcgtgcggaagatacggccgtgtattattgcgcgcgtggtaagggtaatactcataagccttatggttatgttcgttattttgatgtttggggccaaggcaccctggtgacggttagctca (SEQ ID NO: 88) prot VH region ofMS-Roche#7.9H7; protein/1; artificial sequence SEQ ID NO: 89QVQLVESGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAINASGTRTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCARGKGNTHKPYGYVRYFDVWG QGTLVTVSS(SEQ ID NO: 89) VL region of MS-Roche#7.9H7; DNA; artificial sequenceSEQ ID NO: 90Gatatcgtgctgacccagagcccggcgaccctgagcctgtctccgggcgaacgtgcgaccctgagctgcagagcgagccagagcgtgagcagcagctatctggcgtggtaccagcagaaaccaggtcaagcaccgcgtctattaatttatggcgcgagcagccgtgcaactggggtcccggcgcgttttagcggctctggatccggcacggattttaccctgaccattagcagcctggaacctgaagactttgcgacttattattgccttcagatttataatatgcctattacctttggccagggtacgaaagttgaaattaaacgtacg(SEQ ID NO: 90) prot VL region of MS-Roche#7.9H7; protein/1; artificialsequence SEQ ID NO: 91DIVLTQSPATLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGVPARFSGSGSGTDFTLTISSLEPEDFATYYCLQIYNMPITFGQGTKVEIKRT (SEQ ID NO: 91) HCDR3region of MS-Roche#7.9H7; DNA; artificial sequence SEQ ID NO: 92Ggtaagggtaatactcataagccttatggttatgttcgttattttgatgtt (SEQ ID NO: 92) protHCDR3 region of MS-Roche#7.9H7; protein/1; artificial sequence SEQ IDNO: 93 GKGNTHKPYGYVRYFDV (SEQ ID NO: 93) LCDR3 region of MS-Roche#7.9H7;DNA; artificial sequence SEQ ID NO: 94 Cttcagatttataatatgcctatt (SEQ IDNO: 94) prot LCDR3 region of MS-Roche#7.9H7; protein/1; artificialsequence SEQ ID NO: 95 LQIYNMPI (SEQ ID NO: 95)

Further illustrative sequences are depicted in the appended sequencelisting and are also shown in the appended tables, in particular tables1, 8 and 10.

BRIEF DESCRIPTION OF THE DRAWINGS

The Figures show:

FIG. 1 Sequence summary of HuCAL®-Fab1 Library The numbering isaccording to VBASE except the gap in VLλ position 9. In VBASE the gap isset at position 10 (Chothia et al., 1992). In the sequence summary allCDR3 residues which were kept constant are indicated. Correspondingsequences employed for the HuCAL-Fab1 library can be found in theappended sequence listing.

-   -   A: amino acid sequence    -   B: DNA sequence

FIG. 2 Fab display vector pMORPH® 18_Fab Vector map and DNA sequenceincluding restriction sites

FIG. 3 Fab expression vector pMORPH®×9_Fab Vector map and DNA sequenceincluding restriction sites

FIG. 4 Sequences of the parental Fab fragments MS-Roche-3, MS-Roche-7and MS-Roche 8

-   -   A: amino acid sequence    -   B: DNA sequence

FIG. 5: Indirect immunofluorescence of amyloid-plaques from a cryostatsection of human temporal cortex. The plaques were labeled with MS-R#3.2 Fab (upper panels) and MS-R #7.4 Fab (lower panels) at 20 μg/ml(left panels) and 5 μg/ml (right panels) under stringent blockingconditions. Bound MS-R Fab was revealed by goat anti-human-Cy3.

FIG. 6: Indirect immunofluorescence of amyloid-plaques from a cryostatsection of human temporal cortex. The plaques were labeled with MS-R#3.3 IgG1 (upper panels) and MS-R #7.12 IgG1 (lower panels) at 0.05μg/ml (left panels) and 0.01 μg/ml (right panels) under stringentblocking conditions. Bound MS-R IgG1 antibody was revealed by goatanti-human (H+L)-Cy3.

FIG. 7: Indirect immunofluorescence of amyloid-plaques from a cryostatsection of human temporal cortex using antibodies after final affinitymaturation. The plaques were labeled with MS-R #7.9.H7 IgG1 (MAB 31, toppanel), MS-R #7.11.H1×7.2.L1 IgG1 (MAB 11, middle panel) and MS-R#3.4.H7, bottom panel). Antibodies were used at 0.05 μg/ml (left panels)and 0.01 μg/ml (right panels) under stringent blocking conditions. BoundMS-R IgG1 antibody was revealed by goat anti-human (H+L)-Cy3.

-   -   Scale: 8.5 mm=150 μm.

FIG. 8: Polymerization Assay. Anti-Aβ antibodies prevent incorporationof biotinylated Aβ into preformed Aβ aggregates.

FIG. 9: De-polymerization Assay. Anti-Aβ antibodies induce release ofbiotinylated Aβ from aggregated Aβ.

FIG. 10: In vivo decoration of amyloid plaques in an APP/PS2 doubletransgenic mouse after intravenous injection of 1 mg MS-Roche IgG#7.9.H2×7.12.L2. After three days the mouse was perfused withphosphate-buffered saline and sacrificed. The presence of human IgGbound to amyloid plaques was revealed by confocal microscopy afterlabelling cryostat sections from the frontal cortex with a goatanti-human IgG-Cy3 conjugate (panel B). The same section wascounterstained with an anti-Abeta mouse monoclonal antibody(BAP-2-Alexa488 conjugate, panel A) to visualize the position of amyloidplaques. Individual red (panel B) and green (panel A) channels, mergedimage (panel D) and colocalized (pancel C) signals are shown.

-   -   Scale: 1 cm=50 μm

FIG. 11: In vivo decoration of amyloid plaques in an APP/PS2 doubletransgenic mouse after intravenous injection of 1 mg MS-Roche IgG#7.9.H4×7.12.L2. Experimental conditions and staining procedure wereidentical to those described in the legend of FIG. 10.

-   -   Scale: 1.6 cm=50 μm

FIG. 12: In vivo decoration of amyloid plaques in an APP/PS2 doubletransgenic mouse after intravenous injection of 1 mg MS-Roche IgG#7.11.H1×7.2.L1 (MAB 11). Experimental conditions and staining procedurewere identical to those described in the legend of FIG. 10.

-   -   Scale: 1.4 cm=70 μm

FIG. 13: In vivo decoration of amyloid plaques in an APP/PS2 doubletransgenic mouse after intravenous injection of 2 mg MS-Roche IgG#7.9.H7 (MAB 31) at day 0, 3, and 6. After nine days the mouse wasperfused with phosphate-buffered saline and sacrificed. The presence ofhuman IgG bound to amyloid plaques was revealed by confocal microscopyafter labelling cryostat sections from the frontal cortex with a goatanti-human IgG-Cy3 conjugate (panel B). The same section wascounterstained with an anti-Abeta mouse monoclonal antibody(BAP-2-Alexa488 conjugate, panel A) to visualize the position of amyloidplaques. Individual red (panel B) and green (panel A) channels, mergedimage (panel D) and colocalized (panel C) signals and are shown.

-   -   Scale: 1.6 cm=80 μm (panels A, B, C); 1.0 cm=50 μm (panel D)

FIG. 14: In vivo decoration of amyloid plaques in an APP/PS2 doubletransgenic mouse after intravenous injection of 2 mg MS-Roche IgG#7.11.H1×7.2.L1 (MAB 11) at day 0, 3 and 6. Experimental conditions andstaining procedure were identical to those described in the legend ofFIG. 13.

-   -   Scale: 1.6 cm=80 μm

FIG. 15: Binding analysis of anti-Aβ antibodies to cell surface APP.Antibody binding to human APP-transfected HEK293 cells andnon-transfected control cells was analyzed by flow cytometry.

The examples illustrate the invention.

EXAMPLES Example 1 Construction and Screening of a Human CombinatorialAntibody Library (HuCAL®-Fab 1) Cloning of HuCAL®-Fab 1

HuCAL®-Fab 1 is a fully synthetic, modular human antibody library in theFab antibody fragment format. HuCAL®-Fab 1 was assembled starting froman antibody library in the single-chain format (HuCAL®-scFv; Knappik,(2000), J. Mol. Biol. 296, 57-86). Vλ positions 1 and 2. The originalHuCAL® master genes were constructed with their authentic N-termini:VLλ1: QS (CAGAGC), VLλ2: QS (CAGAGC), and VLλ3: SY (AGCTAT). Sequencescontaining these amino acids are shown in WO 97/08320. During HuCAL®library construction, the first two amino acids were changed to DI tofacilitate library cloning (EcoRI site). All HuCAL® libraries containVLX genes with the EcoRV site GATATC (DI) at the 5′-end. All HuCAL®kappa genes (master genes and all genes in the library) contain DI atthe 5′-end (FIGS. 1A and B).

VH position 1. The original HuCAL® master genes were constructed withtheir authentic N-termini: VH1A, VH1B, VH2, VH4, and VH6 with Q (=CAG)as the first amino acid and VH3 and VH5 with E (=GAA) as the first aminoacid. Sequences containing these amino acids are shown in WO 97/08320.During cloning of the HuCAL®-Fab1 library, amino acid at position 1 ofVH was changed to Q (CAG) in all VH genes (FIGS. 1A and B).

Design of the CDR Libraries

Vκ1/Vκ3 position 85. Because of the cassette mutagenesis procedure usedto introduce the CDR3 library (Knappik, (2000), loc. cit.), position 85of Vκ1 and Vκ3 can be either T or V. Thus, during HuCAL®-scFv1 libraryconstruction, position 85 of Vκ1 and Vκ3 was varied as follows: Vκ1original, 85T (codon ACC); Vκ1 library, 85T or 85V (TRIM codons ACT orGTT); Vκ3 original, 85V (codon GTG); Vκ3 library, 85T or 85V (TRIMcodons ACT or GTT); the same applies to HuCAL®-Fab1.

CDR3 design. All CDR3 residues, which were kept constant, are indicatedin FIGS. 1A and B.

CDR3 length. The designed CDR3 length distribution is as follows.Residues, which were varied are shown in brackets (x) in FIG. 1. V kappaCDR3, 8 amino acid residues (position 89 to 96) (occasionally 7-10residues), with Q89, S90, and D92 fixed; and VH CDR3, 5 to 28 amino acidresidues (position 95 to 102) (occasionally 4-28), with D101 fixed.

HuCAL®-Fab 1 was cloned into a phagemid expression vector pMORPH®18_Fab1(FIG. 2). This vector comprises the Fd fragment with a phoA signalsequence fused at the C-terminus to a truncated gene III protein offilamentous phage, and further comprises the light chain VL-CL with anompA signal sequence. Both chains are under the control of the lacoperon. The constant domains Cλ, Cκ and CH1 are synthetic genes fullycompatible with the modular system of HuCAL® (Knappik, (2000), loc.cit.).

The whole VH-chain (MunI/StyI-fragment) was replaced by a 1205 by dummyfragment containing the 8-lactamase transcription unit (bla), therebyfacilitating subsequent steps for vector fragment preparation andallowing for selection of complete VH removal.

After VH-replacement, VLA, was removed by EcoRI/DraIII and VLK byEcoRI/BsIWI and replaced with bacterial alkaline phosphatase (bap) genefragment (1420 bp).

As the variability of the light chains is lower than that of the heavychains, cloning was started with the light chain libraries. The VL_(λ)and VL_(κ) light chain libraries diversified in L-CDR3, which weregenerated for the HuCAL®-scFv library (Knappik, (2000), loc. cit.) werealso used for cloning of HuCAL®-Fab1. In case of λ they consisted of theλ1-, λ2- and λ3-HuCAL®-framework and had a total variability of 5.7×10⁶.VL_(λ) fragments were amplified by 15 PCR cycles (Pwo-polymerase) withprimers 5′-GTGGTGGTTCCGATATC-3′ (SEQ ID NO: 28) and5′-AGCGTCACACTCGGTGCGGCTTTCGGCTGGCCAAGAACGGTTA-3′ (SEQ ID NO: 29).PCR-products were digested with EcoRV/DraIII and gel-purified. In caseof the VL_(λ)-library, the bap-dummy was removed by EcoRV/DraIII fromthe library vector. 2 μg of gel purified vector were ligated with a3-fold molar excess of VL_(λ)-chains for 16 h at 16° C., and theligation mixtures were electroporated in 800 μl E. coli TOP10F cells(Invitrogen), yielding altogether 4.1×10⁸ independent colonies. Thetransformants were amplified about 2000-fold in 2×YT/1% glucose/34 μg/mlchloramphenicol/100 μg/ml ampicillin, harvested and stored in 20% (w/v)glycerol at −80° C.

The κ libraries comprise the κ1-, κ2-, κ3- and κ4-HuCAL® master geneswith a total variability of 5.7×10⁶. VL_(κ)-chains were obtained byrestriction digest with EcoRV/BsIWI and gel-purified. In case of theVL_(κ)-library, the bap-dummy was removed by EcoRV/BsIWI from thelibrary vector. 2 μg of gel-purified vector were mixed with a 5-foldmolar excess of VL_(κ)-chains. Ligation and transformation into E. coliTOP10F cells (Invitrogen) was performed as described for VL_(λ)-chains,yielding altogether 1.6×10⁸ independent colonies. DNA of the two lightchain libraries was prepared and the bla-dummy was removed by MunI/StyI,thereby generating the two vectors for insertion of the VHsub-libraries. The VH libraries of HuCAL®-scFv were used for thegeneration of HuCAL®-Fab1. The VH libraries of HuCAL®-scFv consist ofthe master genes VH1A/B-6 diversified with two VH-CDR3 trinucleotidelibrary cassettes differing in CDR3 length separately, and eachVH-library combined with the VL_(κ)- and with the VL_(λ)-library. Forthe generation of the HuCAL®-Fab1 DNA from these VH-libraries wasprepared preserving the original variability. The DNA was digested withMunI/StyI and gel-purified. A 5-fold molar excess of the VH-chains wasligated with 3 μg of the VL_(λ)-library vector and with 3 μg of theVL_(κ)-library vector for 4 h at 22° C. The ligation mixtures wereelectroporated for each vector in 1200 μl E. coli TOP10F cells(Invitrogen), yielding altogether 2.1×10¹⁰ independent colonies. Thetransformants were amplified about 4000-fold in 2×YT/1% glucose/34 μg/mlchloramphenicol/10 μg/ml tetracycline, harvested and stored in 20% (w/v)glycerol at −80° C. As quality control the light chain and heavy chainof single clones was sequenced with 5″-CAGGAAACAGCTATGAC-3″ (SEQ ID NO:30) and 5″-TACCGTTGCTCTTCACCCC-3″ (SEQ ID NO: 31), respectively.

Phagemid Rescue, Phage Amplification and Purification

HuCAL®-Fab 1 was amplified in 2×TY medium containing 34 μg/mlchloramphenicol, 10 μg/ml tetracycline and 1% glucose (2×TY-CG). Afterhelper phage infection (VCSM13) at 37° C. at an OD₆₀₀ of about 0.5,centrifugation and resuspension in 2×TY/34 μg/ml chloramphenicol/50μg/ml kanamycin cells were grown overnight at 30° C. Phage werePEG-precipitated from the supernatant (Ausubel, (1998), Currentprotocols in molecular biology. John Wiley & Sons, Inc., New York, USA),resuspended in PBS/20% glycerol and stored at −80° C. Phageamplification between two panning rounds was conducted as follows:mid-log phase TG1-cells were infected with eluted phage and plated ontoLB-agar supplemented with 1% of glucose and 34 μg/ml of chloramphenicol.After overnight incubation at 30° C. colonies were scraped off, adjustedto an OD₆₀₀ of 0.5 and helper phage added as described above.

Example 2 Solid Phase Panning

Wells of MaxiSorp™ microtiter plates F96 (Nunc) were coated with 100 μl112.5 μM human Aβ (1-40) peptide (Bachem) dissolved in TBS containingNaN₃ (0.05% v/v) and the sealed plate was incubated for 3 days at 37° C.where the peptide is prone to aggregate on the plate. After blockingwith 5% non-fat dried milk in TBS, 1-5×10¹² HuCAL®-Fab phage purified asabove were added for 1 h at 20° C. After several washing steps, boundphages were eluted by pH-elution with 500 mM NaCl, 100 mM glycin pH 2.2and subsequent neutralisation with 1M TRIS-CI pH 7. Three rounds ofpanning were performed with phage amplification conducted between eachround as described above, the washing stringency was increased fromround to round.

Example 3 Subcloning of Selected Fab Fragments for Expression

The Fab-encoding inserts of the selected HuCAL®-Fab fragments weresubcloned into the expression vector pMORPH®×7_FS to facilitate rapidexpression of soluble Fab. The DNA preparation of the selectedHuCAL®-Fab clones was digested with XbaI/EcoRI, thus cutting out the Fabencoding insert (ompA-VL and phoA-Fd). Subcloning of the purifiedinserts into the XbaI/EcoRI cut vector pMORPH®×7, previously carrying ascFv insert, leads to a Fab expression vector designated pMORPH®×9_Fab1(FIG. 3). Fabs expressed in this vector carry two C-terminal tags (FLAGand Strep) for detection and purification.

Example 4 Identification of Aβ-Binding Fab Fragments by ELISA

Wells of Maxisorp™ microtiter plates F384 (Nunc) were coated with 20 μl112.5 μM human Aβ (1-40) peptide (Bachem) dissolved in TBS containingNaN₃ (0.05% v/v) and the sealed plate was incubated for 3 days at 37°C., where the peptide is prone to aggregate on the plate. Expression ofindividual Fab was induced with 1 mM IPTG for 16 h at 22° C. Soluble Fabwas extracted from E. coli by BEL lysis (boric acid, NaCl, EDTA andlysozyme containing buffer pH 8) and used in an ELISA. The Fab fragmentwas detected with an alkaline phosphatase-conjugated goat anti-Fabantibody (Dianova/Jackson Immuno Research). After excitation at 340 nmthe emission at 535 nm was read out after addition of AttoPhosfluorescence substrate (Roche Diagnostics).

Example 5 Optimization of Antibody Fragments

In order to optimize the binding affinity of the selected Ali bindingantibody fragments, some of the Fab fragments, MS-Roche-3 (MSR-3),MS-Roche-7 (MSR-7) and MS-Roche-8 (MSR-8) (FIG. 4), were used toconstruct a library of Fab antibody fragments by replacing the parentalVL κ₃ chain by the pool of all kappa chains κ1-3 diversified in CDR3from the HuCAL® library (Knappik et al., 2000).

The Fab fragments MS-Roche-3, 7 and 8 were cloned via XbaI/EcoRI frompMORPH®×9_FS into pMORPH®18, a phagemid-based vector for phage displayof Fab fragments, to generate pMORPH®18_Fab1 (FIG. 2). A kappa chainpool was cloned into pMORPH®18_Fab1 via XbaI/SphI restriction sites.

The resulting Fab optimization library was screened by panning againstaggregated human Aβ (1-40) peptide coated to a solid support asdescribed in example 2. Optimized clones were identified by koff-rankingin a Biacore assay as described in Example 8. The optimized clonesMS-Roche-3.2, 3.3, 3.4, 3.6, 7.2, 7.3, 7.4, 7.9, 7.11, 7.12, 8.1, 8.2,were further characterized and showed improved affinity and biologicalactivity compared to the starting fragment MS-Roche-3, MS-Roche-7 andMS-Roche-8 (FIG. 4). The CDRs listed refer to the HuCAL® consensus-basedantibody gene VH3kappa3. The Fab fragment MS-Roche-7.12 was obtained bycloning the HCDR3 of parental clone MS-R 7 into a HuCAL®-Fab library,carrying diversity in all 6 CDR regions using a design procedureidentical with that for CDR3 cassettes described in Knappik et al.,2000. The library cassettes were designed strongly biased for the knownnatural distribution of amino acids and following the concept ofcanonical CDR conformations established by Allazikani (Allazikani etal., 1997). However in contrast to the HuCAL® master genes, the cloneMS-Roche 7.12 contains amino acid Sat position 49 of the VL chain (seeappended table 1).

The optimized Fabs after the first affinity maturation round showedimproved characteristics over the starting MS-Roche-3, MS-Roche-7 andMS-Roche-8 clones (FIG. 4). The binding affinities of the maturated Fabsto A61-40 and A61-42 were significantly increased yielding K_(D) valuesin the range of 22-240 nM in comparison to 850-1714 nM of the parentalclones (Table 3). Immunohistochemistry analysis of amyloid plaques inhuman AD brain sections also showed a significantly increased stainingprofile of the maturated clones, i.e. better signal to background ratioswere obtained and positive plaque staining was detected at relativelylow concentrations of the maturated Fabs (FIG. 5).

For further optimization, the VH CDR2 regions and the VL CDR1 regions ofa set of antibody fragments derived from L-CDR3 optimized MS-Roche-3, -7and -8 (table 1; FIG. 4) were optimized by cassette mutagenesis usingtrinucleotide-directed mutagenesis (Virnekas et al., 1994). Therefore, atrinucleotide-based HCDR2 cassette and a trinucleotide-based LCDR1cassette were constructed using a design procedure identical with thatfor CDR3 cassettes described in Knappik et al., 2000. The librarycassettes were designed strongly biased for the known naturaldistribution of amino acids and following the concept of canonical CDRconformations established by Allazikani (Allazikani et al., 1997). Theprotocol used for the optimization of the initial selected antibodyfragments would mimic the process of affinity maturation by somatichypermutation observed during the natural immune response.

The resulting libraries were screened separately as described aboveleading to optimized clones either in the H-CDR2 or in the L-CDR1region. All clones were identified as above by an improved koff towardsAβ1-40-fibers after a koff-ranking in the Biacore and showed improvedaffinity either to Aβ1-40 or Aβ-42 or both when compared to thecorresponding parent clone (Table 3). Table 1 contains the sequencecharacteristics of the parental as well as sequences of the optimizedclones. The CDRs listed refer to the HuCAL® consensus-based antibodygene VH3kappa3.

For example, the affinity of the MS-Roche-7 parental Fab towards Ab1-40was improved over 35-fold from 1100 nM to 31 nM after L-CDR3optimization (MS-Roche-7.9) and further improved to 5 nM after H-CDR2optimization (MS-Roche-7.9H2) as illustrated in Table 3. The H-CDR2 andL-CDR1 optimization procedure not only increased the affinity but alsoresulted for some of the clones in a significantly improved staining ofamyloid plaques in AD brain section, as particularly seen with MS-Roche7.9H2 and 7.9H3.

TABLE 1 pos. pos. pos. Binder name L-CDR1 49 L-CDR2 85 L-CDR3 H-CDR1 47H-CDR2 H-CDR3 MS-Roche #3 RASQSVSSSYLA Y GASSRAT V QQVYNPPV GFTFSSYAMS WAISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.1 RASQSVSSSYLA Y GASSRAT TQQVYSVPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.2RASQSVSSSYLA Y GASSRAT V QQIYSYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRY FDV MS-Roche #3.3 RASQSVSSSYLA Y GASSRAT V HQMSSYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.4 RASQSVSSSYLAY GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDVMS-Roche #3.5 RASQSVSSSYLA Y GASSRAT T QQIYDYPP GFTFSSYAMS WAISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.6 RASQSVSSSYLA Y GASSRAT VQQTYNYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.2.H1RASQSVSSSYLA Y GASSRAT V QQIYSYPP GFTFSSYAMS W AISEHGLNIYYADSVKGLTHYARYYRY FDV MS-Roche #3.2.H2 RASQSVSSSYLA Y GASSRAT V QQIYSYPPGFTFSSYAMS W AISQRGQFTYYADSVKG LTHYARYYRY FDV MS-Roche #3.3.H1RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W VISEKSRFIYYADSVKGLTHYARYYRY FDV MS-Roche #3.3.H2 RASQSVSSSYLA Y GASSRAT V HQMSSYPPGFTFSSYAMS W VISQESQVKYYADSVKG LTHYARYYRY FDV MS-Roche #3.3.H3RASQSVSSSYLA Y GASSRAT V HQMSSYPP GFTFSSYAMS W AISQNGFHIYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H1 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISETSIRKYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H2RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VIDMVGHTYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H3 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H4RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETGMHIYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H5 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISQVGAHIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H6RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGWSTYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H7 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H8RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISEHGRFKYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H9 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISESSKNKYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H10RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGRGKYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H11 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISEFGKNIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H12RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGQNIYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H13 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISEQGRNIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H14RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISESGQYKYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H16 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISESGVNIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.H17RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISEFGQFIYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.H18 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISQQSNFIYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.L7RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.L8 RASQWITKSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.4.L9RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRY FDV MS-Roche #3.4.L11 RASQLVGRAYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.6.H1RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W VISESGQYKYYADSVKGLTHYARYYRY FDV MS-Roche #3.6.H2 RASQSVSSSYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W VISERGINTYYADSVKG LTHYARYYRY FDV MS-Roche #3.6.H3RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W VISETGKFIYYADSVKGLTHYARYYRY FDV MS-Roche #3.6.H4 RASQSVSSSYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISERGRHIYYADSVKG LTHYARYYRY FDV MS-Roche #3.6.H5RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADSVKGLTHYARYYRY FDV MS-Roche #3.6.H6 RASQSVSSSYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISEHGTNIYYADSVKG LTHYARYYRY FDV MS-Roche #3.6.H8RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISEYSKFKYYADSVKGLTHYARYYRY FDV MS-Roche #3.6.L1 RASQFIQRFYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRY FDV MS-Roche #3.6.L2RASQFLSRYYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRY FDV MS-Roche #7 RASQSVSSSYLA Y GASSRAT T FQLYSDPF GFTFSSYAMSW AISGSGGSTYYADSVKG GKGNTHKPY GYVRYFDV MS-Roche #7.1 RASQSVSSSYLA YGASSRAT V HQLYSSPY GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.3 RASQSVSSSYLA YGASSRAT V HQVYSHPF GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.4 RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.5 RASQSVSSSYLA YGASSRAT T HQVYSSPF GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.6 RASQSVSSSYLA Y GASSRAT V HQLYSPPY GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.7 RASQSVSSSYLA YGASSRAT T HQVYSAPF GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.8 RASQSVSSSYLA Y GASSRAT V HQVYSFPI GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9 RASQSVSSSYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.10 RASQSVSSSYLA Y GASSRAT T QQVYNPPH GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.11 RASQSVSSSYLA YGASSRAT T QQVYSPPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.12 RASQYVSSPYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS WNISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.13 RASQSVSSSYLA YGASSRAT V HQVYSPPF GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2.H1 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAINANGLKKYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.2.H2 RASQSVSSSYLA YGASSRAT T QQIYSFPH GFTFSSYAMS W AINGTGMKKYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2.H3 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAINANGYKTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.2.H4 RASQSVSSSYLA YGASSRAT T QQIYSFPH GFTFSSYAMS W AINSKGSRIYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2.H5 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAINATGRSKYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.2.H6 RASQSVSSSYLA YGASSRAT T QQIYSFPH GFTFSSYAMS W AINARGNRTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2.H7 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAINSRGSDTHYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.2.H8 RASQSVSSSYLA YGASSRAT T QQIYSFPH GFTFSSYAMS W AINASGHKTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2.L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.2.L2 RASQYISFRYLA YGASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.2.L4 RASQFIRRSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.3.H1 RASQSVSSSYLA YGASSRAT V HQVYSHPF GFTFSSYAMS W AISAISNKTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.3.L1 RASQYLHYGYLA Y GASSRAT V HQVYSHPF GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.4.H1 RASQSVSSSYLA YGASSRAT V QQIYNFPH GFTFSSYAMS W AINATGYRTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.4.H2 RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS WAINYNGARIYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9.H1 RASQSVSSSYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AINANGQRKFYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.9.H2 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS WAINADGNRKYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9.H3 RASQSVSSSYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AINYQGNRKYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.9.H4 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS WAINAVGMKKFYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9.H5 RASQSVSSSYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AINHAGNKKYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.9.L1 RASQRLSPRYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS WAISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9.L2 RASQYLHKRYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.9.H6 RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WAINARGNRTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9.H7 RASQSVSSSYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AINASGTRTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.9.H8 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS WAINASGSKIYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.9.H9 RASQSVSSSYLA YGASSRAT T LQIYNMPI GFTFSSYAMS W AINGKGNKKYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.11.H1 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WGINAAGFRTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.11.H2 RASQSVSSSYLA YGASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.11.H3 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WGINANGNRTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.11.H4 RASQSVSSSYLA YGASSRAT T QQVYSPPH GFTFSSYAMS W AINANGYKTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.11.H5 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WAINAHGQRTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.11.L1 RASQRILRIYLA YGASSRAT T QQVYSPPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.12.H1 RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS WNINGNGNRKYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.12.L1 RASQYVFRRYLA SGSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.12.L2 RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS WNISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.12.L3 RASQFVRRGFLA SGSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.12.L4 RASQRLKRSYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS WNISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.12.L5 RASQRLKRSYLA SGSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #7.12.L6 RASQYLWYRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS WNISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDV MS-Roche #7.12.L7 RASQWIRKTYLA SGSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKP YGYVRYFDVMS-Roche #8 RASQSVSSSYLA Y GASSRAT T QQLSSFPP GFTFSSYAMS WAISGSGGSTYYADSVKG LLSRGYNGY YHKFDV MS-Roche #8.1 RASQSVSSSYLA Y GASSRATT QQLSNYPP GFTFSSYAMS W AISGSGGSTYYADSVKG LLSRGYNGY YHKFDV MS-Roche #8.2RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLLSRGYNGY YHKFDV MS-Roche #8.1.H1 RASQSVSSSYLA Y GASSRAT T QQLSNYPPGFTFSSYAMS W AISRSGSNIYYADSVKG LLSRGYNGY YHKFDV MS-Roche #8.2.H1RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISITGRRKYYADSVKGLLSRGYNGY YHKFDV MS-Roche #8.2.H2 RASQSVSSSYLA Y GASSRAT T QQLSSYPPGFTFSSYAMS W AISRTGSKTYYADSVKG LLSRGYNGY YHKFDV MS-Roche #8.2.H4RASQSVSSSYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W ATSVKGKTYYADSVKGLLSRGYNGY YHKFDV MS-Roche #8.2.L1 RASQRVSGRYLA Y GASSRAT T QQLSSYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LLSRGYNGY YHKFDV

Sequences belonging to V_(H)3 and Vκ3 HuCAL consensus sequences see FIG.1A

Example 6 Construction of HuCAL® Immunoglobulin Expression Vectors

Heavy chain cloning. The multiple cloning site of pcDNA3.1+ (invitrogen)was removed (NheI/ApaI), and a stuffer compatible with the restrictionsites used for HuCAL® design was inserted for the ligation of the leadersequences (NheI/EcoRI), VH-domains (MunI/), and the immunoglobulinconstant regions (BlpI/ApaI). The leader sequence (EMBL 83133) wasequipped with a Kozak sequence (Kozak, 1987). The constant regions ofhuman IgG (PIR A02146), IgG4 (EMBL K01316), and serum IgA1 (EMBL J00220)were dissected into overlapping oligonucleotides with length of about 70bases. Silent mutations were introduced to remove restriction sitesnon-compatible with the HuCAL® design. The oligonucleotides were splicedby overlap extension-PCR.

During sub-cloning from Fab into IgG, the VH DNA sequence of the Fab iscut out via Mfe I/Blp I and ligated into the IgG vector opened via EcoRI/Blp I. EcoR I (g/aattc) and Mfe I (c/aattg) share compatible cohesiveends (aatt) and the DNA sequence of the original Mfe I site in the Fabchanges from: c/aattg to: g/aattg after ligation into the IgG expressionvector, thereby destroying both Mfe I and EcoR I site, and thus alsoleading to an amino acid change from Q (codon: caa) to E (codon: gaa).The V_(H) DNA sequence of the IgG of antibody molecule 7.9H7 aftersubcloning is shown in SEQ ID No.: 424, and the corresponding amino acidsequence is shown in SEQ ID No: 425.

Light chain cloning. The multiple cloning site of pcDNA3.1/Zeo+(Invitrogen) was replaced by two different stuffers. The K-stufferprovided restriction sites for insertion of a κ-leader (NheI/EcoRV),HuCAL®-scFv Vκ-domains (EcoRV/BsiWI), and the κ-chain constant region(BsiWI/ApaI). The corresponding restriction sites in the λ-stuffer wereNheI/EcoRV (λ-leader), EcoRV/HpaI (Vλ-domains), and HpaI/ApaI (λ-chainconstant region). The κ-leader (EMBL Z00022) as well as the λ-leader(EMBL J00241) were both equipped with Kozak sequences. The constantregions of the human κ-(EMBL L00241) and λ-chain (EMBL M18645) wereassembled by overlap extension-PCR as described above.

Generation of IgG-expressing CHO-cells. CHO-K1 cells were co-transfectedwith an equimolar mixture of IgG heavy and light chain expressionvectors. Double-resistant transfectants were selected with 600 μg/mlG418 and 300 μg/ml Zeocin (Invitrogen) followed by limiting dilution.The supernatant of single clones was assessed for IgG expression bycapture-ELISA. Positive clones were expanded in RPMI-1640 mediumsupplemented with 10% ultra-low IgG-FCS (Life Technologies). Afteradjusting the pH of the supernatant to 8.0 and sterile filtration, thesolution was subjected to standard protein A column chromatography(Poros 20 A, PE Biosystems).

Example 7 Pepspot Analysis with Decapeptides

The following amino acid sequence encompassing Aβ (1-42) was dividedinto 43 overlapping decapeptides with a frameshift of 1 amino acid.ISEVKM¹DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGWI⁴²ATV IV (SEQ ID NO:414). Accordingly, DAEF RHDSGYEVHH QKLVFFAEDV GSNKGAIIGL MVGGWIA (SEQ IDNO: 27) as enclosed represents amino acids 1 to 42 of Aβ4/β-A4 peptide.

The 43 decapeptides were synthesized with N-terminal acetylation andC-terminal covalent attachment to a cellulose sheet (“pepspot”) by acommercial supplier (Jerini BioTools, Berlin). The cellulose sheet isincubated for 2 hours on a rocking platform with monoclonal antibody (2μg/ml) in blocking buffer (50 mM Tris.HCl, 140 mM NaCl, 5 mM NaEDTA,0.05% NP40 (Fluka), 0.25% gelatine (Sigma), 1% bovine serum albuminefraction V (Sigma), pH 7.4). The sheet is washed 3 times 3 minutes on arocking platform with TBS (10 mM Tris.HCl, 150 mM NaCl, pH 7.5). It isthen wetted with cathode buffer (25 mM Tris base, 40 mM 6-Aminohexaneacid, 0.01% SDS, 20% methanol) and transferred to a semi-dry blottingstack with the peptide side facing a PVDF membrane (Biorad) of equalsize.

The semi-dry blotting stack consists out of freshly wetted filter papers(Whatman No. 3) slightly larger than the peptide sheet:

3 papers wetted with Cathode bufferthe peptide sheeta sheet of PVDF membrane wetted with methanol3 papers wetted with Anode buffer 1 (30 mM Tris base, 20% methanol)3 papers wetted with Anode buffer 2 (0.3 mM Tris base, 20% methanol)

The transfer is conducted at a current density between Cathode and Anodeof 0.8 mA/cm² for 40 minutes which is sufficient to elute most of theantibody from the cellulose sheet and deposit it on the PVDF membrane.The PVDF membrane is then exchanged for a 2^(nd) PVDF membrane andtransferred for another 40 minutes to ensure complete elution from thecellulose sheet.

The PVDF membrane is immersed in blocking buffer for 10 minutes. ThenHRP-labeled anti-human Ig H+L (Pierce) is added at 1:1000 dilution andthe membrane is incubated on a rocking platform for 1 hour. It is washed3×10 minutes with TBST (TBS with 0.005% Tween20). Color is developed byimmersing the membrane into a solution made of 3 mg 4-chloronaphtholdissolved in 9 ml methanol with 41 ml PBS (20 mM Na-phosphate, 150 mMNaCl, pH 7.2) an 10 μl 30% hydrogen peroxide (Merck). After thedevelopment of blue-black spots the membrane is washed extensively withwater and dried.

The assignment of antibody-reactive pepspots is made by visualinspection through a transparent spot matrix. The epitopes of theantibody in question is defined as the minimal amino acid sequence inreactive peptides. For comparison mouse monoclonal antibodies (BAP-2,BAP-1, BAP-17 BAP-21, BAP-24, and 4G8) are analyzed in the same way,except using HRP-labeled anti-mouse Ig instead of anti-human Ig.

It is of note that affinity maturation and conversion of the monovalentFab fragments into full-length IgG1 antibodies results usually in somebroadening of the epitope recognition sequence as indicated by pepspotand ELISA analyses. This may be related to the recruitment of morecontact points in the antibody-antigen interaction area as a consequenceof the affinity maturation or to a stronger binding to the minimalepitope such that also weak interactions with adjacent amino acid can bedetected. The latter may be the case when Aβ-derived peptides are probedwith full-length IgG antibodies. As illustrated in Table 2 for thepepspot analysis, the recognition sequences of the N-terminal and middleepitopes are extended by up to three amino acids when parent Fabs andcorresponding fully maturated IgG antibodies are compared. However, ithas to be kept in mind that the decapeptides are modified for covalentattachment at the C-terminal amino acid and this amino acid maytherefore not easily be accessible to the full-length antibody due tosteric hindrance. If this is the case the last C-terminal amino aciddoes not significantly contribute to the epitope recognition sequenceand a potential reduction of the minimal recognition sequence by oneamino acid at the C-terminal end has to be considered in the pepspotanalysis as used in the present invention.

TABLE 2 Pepspot analysis of binding Fabs and full-length IgG antibodiesto decapeptides on a cellulose sheet. The numbers refer to the essentialamino acids from the Aβ1-40 sequence which have to be present in thedecapeptide for optimal binding of antibody. A weak peptide reactivity,and hence a weak contribution to the epitope, is indicated by brackets.antibody position position MSR-3 Fab 3-4 18-23 MSR-7 Fab 3-5 19-24 MSR-8Fab 4-5 18-21 MSR-9 Fab (1)3-9 18-24 MSR-10 Fab  (4-10) 19-20 MSR-11 Fab3-7 (18-20) MSR-26 Fab 3-5 (16)-19-23 MSR-27 Fab (3)6-9 13-18(20) MSR-29Fab 14-16(20) MSR-37 Fab (4-6) (19-24) MSR-41 Fab 3-7 (17-21) MSR-42 Fab(4-9) (18-24) MSR 3.4.H7 IgG1 1-3 19-26 MSR 7.9.H2 IgG1 1-4 19-24 MSR7.9.H7 IgG1 4-6 19-26 MSR 7.2.H2 × 7.2.L1 IgG1 (1-4) 18-26 5-9 MSR7.11.H1 × 7.2.L1 IgG1 4-6 19-26 BAP-2 4-6 4G8 19-20(23) BAP-21 32-34BAP-24 38-40 BAP-1 4-6 BAP-17 38-40

Table 2: Pepspot analysis of binding Fabs and full-length IgG antibodiesto decapeptides on a cellulose sheet. The numbers refer to the essentialamino acids from the Aβ1-40 sequence which have to be present in thedecapeptide for optimal binding of antibody. A weak peptide reactivity,and hence a weak contribution to the epitope, is indicated by brackets

Example 8 Determination of K_(D) Values for MS-R Fab and MS-R IgG1Antibody Binding to Aβ1-40 and Aβ1-42 Fibers in Vitro by Surface PlasmonResonance (SPR)

Binding of anti-Aβ antibodies (Fabs and IgG1) to fibrillar Aβ wasmeasured online by surface plasmon resonance (SPR), and the affinitiesof the molecular interactions were determined as described by Johnson,Anal. Biochem. 1991, 198, 268-277, and Richalet-Sécordel, Anal. Biochem.1997, 249, 165-173. Biacore2000 and Biacore3000 instruments were usedfor these measurements. Aβ1-40 and Aβ1-42 fibers were generated in vitroby incubation of synthetic peptides at a concentration of 200 μg/ml in10 mM Na-acetate buffer (pH 4.0) for three days at 37° C. Electronmicroscopic analysis confirmed a fibrillar structure for both peptides,Aβ1-40 showing predominantly shorter (<1 micron) and Aβ1-42predominantly longer (>1 micron) fibers. These fibers are assumed torepresent aggregated Aβ peptides in human AD brain more closely thanill-defined mixtures of amorphous aggregates and unstructuredprecipitates. The fibers were diluted 1:10 and directly coupled to a“Pioneer Sensor Chip F1” as described in the Instruction Manual of themanufacturer (BIAapplication Handbook, version AB, Biacore AB, Uppsala,1998). In initial experiments it was found that selected MS-Roche Fabsdiffered substantially in their reaction kinetics and therefore the modeof data analysis had to be chosen accordingly. For binders with slowkinetics K_(D) values were calculated by curve fitting of thetime-dependent sensor responses, i.e. from the ratio of k_(off)/k_(on).Binders with fast kinetics were analyzed by fitting theconcentration-dependent sensor responses at equilibrium(adsorption-isotherms). K_(D) values were calculated from the Biacoresensograms based on the total Fab concentration as determined by aprotein assay. For the clones derived from the 1^(st) and 2^(nd)affinity maturation cycle the content of active Fab in each preparationwas determined in the Biacore according to a method described byChristensen, Analytical Biochemistry (1997) 249, 153-164. Briefly,time-dependent protein binding to Aβ1-40 fibers immobilized on theBiacore chip was measured during the association phase undermass-limited conditions at different flow rates of the analyte solution.The conditions of mass limitation were realized by immobilizing highamounts of Aβ fibers (2300 response units) on the chip surface of ameasuring channel and by working at relatively low analyteconcentrations, i.e. 160 nM (based on the total Fab proteinconcentration).

A summary of the K_(D) values of selected MS-Roche clones identified inthe primary screen of the HuCAL library and their correspondingmaturated derivatives after the 1^(st) and 2^(nd) affinity maturationcycle is shown in Table 3. In the 1^(st) affinity maturation cycle theheavy chain CDR3 (VH-CDR3) was kept constant and optimization wasfocussed on diversification of the light chain CDR3 (VL-CDR3). In the2^(nd) affinity cycle diversification of VL-CDR1 and VH-CDR2 wasperformed. Some of the binders from the 1^(st) maturation cycle wereconverted to full-length human IgG1 antibodies according to thetechnology developed by MorphoSys as described in Example 6 and K_(D)values determined in the Biacore as described above. The K_(D) valuesfor full-length IgG1 binding to Aβ1-40 and Aβ1-42 fibers are shown inTable 4.

Matured derivatives from both the L-CDR1 as well as H-CDR2 library afterthe 2^(nd) maturation cycle were identified and allowed combination oflight and heavy chains. The cross-cloning strategy is described inExample 13. Either whole light chains, LCDR1 or L-CDR1+2 were exchanged.K_(D) values of selected cross-cloned Fabs are shown in Table 8. Some ofthe Fabs from the 1^(st) and 2^(nd) maturation cycles and from thecross-cloned binders were converted to full-length human IgG1 antibodiesaccording to the technology developed by MorphoSys as described inExample 6. K_(D) values of IgG binding to Aβ1-40 and Aβ1-42 fibers weredetermined in the Biacore. Briefly, a kinetic model for the stepwiseformation of a bivalent complex was used, and K_(D) values werecalculated by Scatchard type analysis of equilibrium binding. Due to thevery slow association process at low antibody concentration (severalhours to reach equilibrium) equilibrium binding data were obtained byextrapolation of the association curves to long time intervals. The on-and off rates for the formation of the monovalent and bivalent complexwere determined via the curve fit procedure and used for theextrapolation. Based on these R_(eq) values a Scatchard analysis wasperformed and K_(D) values for the formation of the monovalent and thebivalent complex were determined. The data are summarized in Table 5.From the curvilinear Scatchard plot a higher (bivalent) and lower(monovalent) affinity interaction was derived for the MS-R IgGs derivedfrom the 2^(nd) affinity maturation cycle and cross-clones. These twoaffinities represent the lower and upper K_(D) values of the rangeindicated in Table 5.

TABLE 3 Table 3: K_(D) values for MS-R Fab binding to Aβ1-40 and Aβ1-42fibers as determined in the Biacore. For the clones derived from the1^(st) and 2^(nd) affinity maturation cycle the values are corrected forthe content of active Fab present in each sample as described in thetext. K_(D) Aβ₁₋₄₀ K_(D) Aβ₁₋₄₂ K_(D) Aβ₁₋₄₀ K_(D) Aβ₁₋₄₂ K_(D) Aβ₁₋₄₀K_(D) Aβ₁₋₄₂ Secreted clones from M S-R # nM nM M S-R # nM nM M S-R # nMnM primary screen 3 930 1300 7 1100 1714 8 850 1000 1^(st) affinitymaturation 3.2 52 240 7.2 22 58 8.1 24 42 3.3 38 104 7.3 23 88 8.2 24 643.4 32 103 7.4 28 103 3.6 40 68 7.9 31 93 7.11 22 74 7.12 28 60 2^(nd)affinity maturation 3.2H1 4.4 3.3 7.2H1 9.3 10.2 8.1H1 13.6 9.2 3.2H25.2 1.1 7.2H2 8.2 8.2 8.2H1 1.6^(a) 2.1^(a) 3.3H1 17.1 19.4 7.2H3 45.45.3 8.2H3 n.d. 3.1 3.3H2 10.6 22.8 7.2H4 5.9 5.0 8.2H4 12.1 11.9 3.3H31.4 3.3 7.2H5 8.0 10.1 8.2L1 4.8 3.7 3.4H1 13.5 14.0 7.2H6 1.0 n.d.3.4H3 6.7 8.4 7.2H7 15.5 8.1 3.4H4 33.0 43.0 7.2H8 1.5 2.1 3.4H5 26.536.0 7.2L1 13.3 12.7 3.4H6 49.0 60.0 7.2L2 5.6 4.0 3.4H7 19.2 31.7 7.2L41.1 1.1 3.4H8 10.7 26.5 7.3H1 8.0 11.2 3.4H9 21.7 18.6 7.3L1 4.5 6.03.4H10 8.1 10.1 7.4H1 8.0 6.6 3.4H11 19.5 8.3 7.4H2 9.9 6.2 3.4H12 25.527.0 7.9H1 4.9 5.4 3.4H13 32.3 18.8 7.9H2 5.0 5.7 3.4H14 13.3 16.8 7.9H34.2 2.8 3.4H16 25.5 15.6 7.9H4 4.8 4.2 3.4H17 2.0 4.3 7.9H5 1.7 1.83.4H18 17.1 10.0 7.9H6 1.2 1.2 3.4L7 9.3 9.3 7.9H7 1.0 0.9 3.4L8 6.213.0 7.9H8 0.8 0.7 3.4L9 16.3 9.1 7.9H9 0.9 0.9 3.4L11 5.3 2.6 7.9L1 1.01.1 3.6H1 18.9 23.1 7.9L2 1.0 0.5 3.6H2 19.8 54.0 7.11H1 12.7 6.7 3.6H35.4 7.5 7.11H2 0.3 0.3 3.6H4 13.0 7.8 7.11H3 6.6 4.4 3.6H5 8.2 6.07.11H4 1.0 1.7 3.6H6 36.0 11.8 7.11H5 3.4 1.7 3.6H8 2.5 2.5 7.11L1 1.11.2 3.6L1 15.6 11.1 7.12H1 0.6 0.8 3.6L2 13.7 13.1 7.12L1 n.d. 3.87.12L2 4.0 5.4 7.12L3 0.8 0.9 7.12L4 2.0 0.6 7.12L5 0.8 0.6 7.12L6 n.d.n.d. 7.12L7 n.d. n.d. ^(a)values were calculated from theconcentration-dependent sensor responses at equilibrium; n.d., notdetermined.

TABLE 4 Table 4: K_(D) values for MS-R IgG1 binding to Aβ1-40 and Aβ1-42fibers as determined in the Biacore. The IgGs were derived from MS-RFabs selected after the 1^(st) affinity maturation cycle. The values arecorrected for the content of active MS-R IgGs present in each sample asdescribed in the text. K_(D) Aβ₁₋₄₀ K_(D) Aβ₁₋₄₂ MS-R # nM nM  3.3 IgG13.7 6.6 7.11 IgG1 2.3 5.7 7.12 IgG1 3.1 13.7  8.1 IgG1 6.6 12.3

TABLE 5 K_(D) values for MS-R IgG1 binding to Aβ1-40 and Aβ1-42 fibersas determined in the Biacore. The IgGs were derived from MS-R Fabsselected after the 1^(st) and 2^(nd) affinity maturation cycle and fromcrosscloned Fabs. The values are corrected for the content of activeMS-R IgGs present in each sample as described in the text. The two K_(D)values given for MS-R IgGs derived from the 2^(nd) affinity maturationstep and cross-cloned binders represent higher and lower affinityinteraction as calculated from the curvilinear Scatchard plots. With anumber of additional MS-R IgGs (for example MS-R IgG 7.9.H2 × 7.12.L2and MS-R IgG 7.9.H4 × 7.12.L2), complex curvilinear Scatchard blots wereobtained and determination of K_(D)-values was therefore not possible.K_(D) Aβ₁₋₄₀ K_(D) Aβ₁₋₄₂ Selected clones from MS-R IgG1 nM nM 1^(st)affinity maturation 3.3 3.7 6.6 7.11 2.3 5.7 7.12 3.1 13.7  8.1 6.612.3  2^(nd) affinity maturation 3.4.H7 0.10-0.30 0.10-0.30 7.2.H40.09-0.30 0.10-0.66 7.9.H2 0.12-0.42 0.11-0.38 7.9.H3 0.10-0.500.10-0.40 7.9.H7 0.25-0.69 0.24-0.70 7.12.L1 1.20-3.50 0.74-2.90 8.2.H20.16-1.00 0.12-0.92 cross-cloned Fabs 3.6.H5 × 3.6.L2 0.20-1.030.20-0.95 3.6.H8 × 3.6.L2 0.22-0.95 0.22-0.82 7.4.H2 × 7.2.L1 0.12-0.630.12-0.56 7.11.H1 × 7.2.L1 0.14-0.66 0.15-0.67 7.11.H1 × 7.11.L10.11-0.70 0.13-0.70

Example 9 Staining of Genuine Human Amyloid Plaques in Brain Sections ofan Alzheimer's Disease Patient by Indirect Immunofluorescence

Selected MS-Roche Fabs and full-length IgG1 were tested for binding toβ-amyloid plaques by immunohistochemistry analysis. Cryostat sections ofunfixed tissue from human temporal cortex (obtained postmortem from apatient that was positively diagnosed for Alzheimer's disease) werelabeled by indirect immunofluorescence using MS-Roche Fabs orfull-length human IgG1 antibodies at various concentrations. Fabs andIgG1 antibodies were revealed by goat anti-human affinity-purifiedF(ab′)₂ fragment conjugated to Cy3 and goat anti-human (H+L) conjugatedto Cy3, respectively. Both secondary reagents were obtained from JacksonImmuno Research. Controls included an unrelated Fab and the secondaryantibodies alone, which all gave negative results. Typical examples ofplaque stainings with selected MS-Roche Fabs and MS-Roche IgG1antibodies are shown in FIGS. 5 to 7.

Example 10 Polymerization Assay: Prevention of AR Aggregation

Synthetic Aβ when incubated in aqueous buffer over several daysspontaneously aggregates and forms fibrillar structures which aresimilar to those seen in amyloid deposits in the brains of Alzheimer'sDisease patients. We have developed an in vitro assay to measureincorporation of biotinylated Aβ into preformed A13 aggregates in orderto analyze the Aβ-neutralizing potential of anti-Aβ antibodies and otherAβ-binding proteins such as albumin (Bohrmann et al., 1999, J. Biol.Chem. 274, 15990-15995). The effect of small molecules on Aβ aggregationcan also be analyzed in this assay.

Experimental Procedure

NUNC Maxisorb microtiter plates (MTP) are coated with a 1:1 mixture ofAβ1-40 and Aβ1-42 (2 μM each, 100 μl per well) at 37° C. for three days.Under these conditions highly aggregated, fibrillar Aβ is adsorbed andimmobilized on the surface of the well. The coating solution is thenremoved and the plates are dried at room temperature for 2-4 hours. (Thedried plates can be stored at −20° C.). Residual binding sites areblocked by adding 300 μl/well phosphate-buffered saline containing 0.05%Tween 20 (T-PBS) and 1% bovine serum albumin (BSA). After 1-2 hoursincubation at room temperature the plates are washed 1× with 300 μlT-PBS. A solution of 20 nM biotinylated Aβ1-40 in 20 mM Tris-HCl, 150 mMNaCl pH 7.2 (TBS) containing 0.05% NaN₃ and serially diluted antibody isadded (100 μl/well) and the plate incubated at 37° C. overnight. Afterwashing 3× with 300 μl T-PBS a streptavidin-POD conjugate (RocheMolecular Biochemicals), diluted 1:1000 in T-PBS containing 1% BSA, isadded (100 μl/well) and incubated at room temperature for 2 hours. Thewells are washed 3× with T-PBS and 100 μl/well of a freshly preparedtetramethyl-benzidine (TMB) solution are added. [Preparation of the TMBsolution: 10 ml 30 mM citric acid pH 4.1 (adjusted with KOH)+0.5 ml TMB(12 mg TMB in 1 ml acetone+9 ml methanol)+0.01 ml 35% H₂O₂]. Thereaction is stopped by adding 100 μl/well 1 N H₂SO₄ and absorbance isread at 450 nm in a microtiter plate reader.

Result:

FIG. 8 shows that MS-Roche IgG1 antibodies prevented incorporation ofbiotinylated 40 into preformed Aβ1-40/Aβ1-42 aggregates. TheAβ-neutralizing capacity of these full-length human IgGs was similar tothat of the mouse monoclonal antibody BAP-1 which had been generated bya standard immunization procedure and specifically recognizes amino acidresidues 4-6 of the Aβ peptide when analyzed by the Pepspot technique asdescribed in example 7. Mouse monoclonal antibody BAP-2 which alsoreacts exclusively with amino acids 4-6 (Brockhaus, unpublished) wassignificantly less active in this assay. An even lower activity wasfound with the Aβ1-40 C-terminal specific antibody BAP-17 (Brockhaus,Neuroreport 9 (1998), 1481-1486) and the monoclonal antibody 4G8 whichrecognizes an epitope between position 17 and 24 in the Aβ sequence(Kim, 1988, Neuroscience Research Communication Vol. 2, 121-130). BSA ata concentration of up to 10 μg/ml did not affect incorporation ofbiotinylated Aβ and served as a negative control. However, at higherconcentrations, i.e. >100 μg/ml, BSA has been reported to inhibitbinding of biotinylated Aβ into preformed Aβ fibers (Bohrmann, (1999) JBiol Chem 274 (23), 15990-5) indicating that the interaction of BSA withAβ is not of high affinity.

Example 11 De-Polymerization Assay: Release of Biotinylated Aβ fromAggregated Aβ

In a similar experimental setup we have tested the potential of MS-RocheIgG antibodies to induce depolymerization of aggregated A3. BiotinylatedAβ1-40 was first incorporated into preformed Aβ1-40/Aβ1-42 fibers beforetreatment with various anti-Aβ antibodies. Liberation of biotinylated Aβwas measured using the same assay as described in the polymerizationassay.

Experimental Procedure:

NUNC Maxisorb microtiter plates (MTP) are coated with a 1:1 mixture ofAβ1-40 and Aβ1-42 as described in the polymerization assay. Forincorporation of biotinylated Aβ the coated plates are incubated with200 μl/well 20 nM biotinylated Aβ1-40 in TBS containing 0.05% NaN₃ at37° C. overnight. After washing the plate with 3×300 μl/well T-PBS,antibodies serially diluted in TBS containing 0.05% NaN₃ were added andincubated at 37° C. for 3 hours. The plate was washed and analyzed forthe presence of biotinylated A131-40 as described above.

Result:

FIGS. 9A to D shows that the inventive antibodies inducedde-polymerization of aggregated Aβ as measured by the release ofincorporated biotinylated Aβ1-40. The MS-R antibodies and the mousemonoclonal antibody BAP-1 were similarly active whereas the BAP-2,BAP-17 and 4G8 antibodies were clearly less efficient in liberatingbiotinylated Aβ from the bulk of immobilized Aβ aggregates. BAP-1 canclearly be differentiated from the MS-R antibodies by its reactivitywith cell surface full-length APP (see FIG. 15), and antibodies likeBAP-1 with such properties are not useful for therapeutic applicationsas potential autoimmunological reactions may be induced. It isinteresting to note that BAP-2, despite its specificity for amino acidresidue 4-6 which is exposed in aggregated Aβ has a clearly loweractivity in this assay indicating that not all N-terminus specificantibodies a priori are equally efficient in releasing Aβ from preformedaggregates. The MS-Roche IgGs are clearly superior to BAP-2 with respectto the depolymerizing activity. The relatively low efficiency of BAP-17(C-terminus-specific) and 4G8 (amino acid residues 16-24-specific) inthis assay is due to the cryptic nature of these two epitopes inaggregated A. As already noted in the polymerization assay, BSA at theconcentrations used here had no effect on aggregated Aβ.

The MS-R antibodies derived from the 2^(nd) affinity maturation cycleand from the cross-cloned binders show in general a higher efficacy inthe de-polymerization assay (comparison of FIG. 9A with FIGS. 9B and C),which is consistent with the increased binding affinity of theseantibodies (see tables 3-5). The monoclonal antibodies AMY-33 and 6F/3Dhave been reported to prevent Aβ aggregation in vitro under certainexperimental conditions (Solomon, (1996) Proc. Natl. Acad. Sci. USA 93,452-455; AMY-33 and 6F/3D antibodies were obtained from ZymedLaboratories Inc., San Francisco (Order No. 13-0100) and DakoDiagnostics AG, Zug, Switzerland (Order No. M087201), respectively). Asdemonstrated in FIG. 9D both of these antibodies were completelyinactive in the de-polymerization assay.

Example 12 Epitope Analysis by ELISA on Peptide Conjugates

The following heptapeptides (single letter code) were obtained bysolid-phase synthesis and purified by liquid chromatography using thetechniques known in the art.

AEFRHDC EFRHDSC FRHDSGC RHDSGYC HDSGYEC DSGYEVC SGYEVHC YEVHHQC EVHHQKCVHHQKLC HHQKLVC HQKLVFC QKLVFFC KLVFFAC LVFFAEC VFFAEDC FFAEDVC FAEDVGCAEDVGSC EDVGSNC DVGSNKC VGSNKGC GSNKGAC CSNKGAI CNKGAII CKGAIIG CGLMVGGCMVGGVV CGGVVIA

The peptides were dissolved in DMSO to arrive at 10 mM concentration.

Bovine Albumin (essentially fatty acid free BSA, Sigma Lot 112F-9390)was dissolved to 10 mg/ml in 0.1M sodium bicarbonate and activated byaddition per ml of 50 μl of a 26 mg/ml solution ofN-succinmidyl-maleinimido propionate (NSMP, Pierce) in DMSO. After 15minutes reaction at room temperature the activated BSA was purified bygel filtration (NAP-10, Pharmacia) in PBS with 0.1% sodium azide assolvent. 50 μl of NSMP activated BSA (6.7 mg/ml) was diluted with 50 μlof PBS, 0.1% sodium azide and 10 μl of peptide solution (1 mM in DMSO)was added. As negative control activated BSA was mock-treated withoutpeptide addition. After 4 hrs at room temperature the reaction wasstopped by addition of 10 μl of 10 mM Cystein. An aliquot of theconjugate reaction mixture was diluted 1:100 with 0.1M sodiumbicarbonate buffer and immediately filled into the wells (100 μl) ofELISA plates (Nunc Immuno-Plate). After standing 16 hrs at 4° C. 100 μlblocking buffer (as above) was added to each well and incubated foranother 30 minutes. The plates were washed with 2×300 μl/well TBST (asabove) and filled with 100 μl antibody at 10 μg/ml or 2 μg/ml inblocking buffer. The plates were kept 16 hours at 4° C. and washed with2×300 μl TBST. 100 μl/well HRP-conjugated anti-human Ig H+L (Pierce,dilution 1:1000 with blocking buffer) was added and incubated for 1 hourat ambient temperature. The plates were washed with 3×300 μl/well TBST.Colour development was started by addition of 100 μl tetra-methylbenzidine/hydrogen peroxide reagent. The reaction was stopped after 5minutes by addition of 100 μl/well 1M sulfuric acid and the opticaldensity is measured by an optical reader (Microplate Reader 3550,BioRad) at 450 nm. For comparison mouse monoclonal antibodies wereanalysed in the same way, except using as revealing agent HRP-labelledanti-mouse Ig instead of anti-human Ig.

Employing specific of the above described heptapeptides derived from AR,specific ELISA-tests as described herein above were carried out.Preferably, inventive antibodies comprise antibodies which show, asmeasured by of optical densities, a signal to background ratio above“10” when their reactivity with an A-beta derived peptide (AEFRHD, SEQID NO: 415; amino acid 2 to 7 of A-beta) is compared to an non-relatedprotein/peptide like BSA. Most preferably, the ratio of opticaldensities is above “5” for a corresponding reaction with at least one ofthe following three Aβ derived peptides: (VFFAED, SEQ ID NO: 421; aminoacid 18 to 23 of Aβ) or (FFAEDV, SEQ ID NO: 423; amino acid 19 to 24 ofAβ) or (LVFFAE, SEQ ID NO: 420; amino acid 17 to 22 of Aβ).

Corresponding results for the inventive parental and/or maturatedantibodies are shown in the following two tables:

TABLE 6 Reactivity of MS-R Fabs with BSA-conjugated Abeta heptapeptides2-7 (AEFRHD, SEQ ID NO: 415), 17-22 (LVFFAE, SEQ ID NO: 420), 18-23(VFFAED, SEQ ID NO: 421) and 19-24 (FFAEDV, SEQ ID NO: 423). The ratiosof the ELISA read-out (optical density) obtained with peptide-conjugatedand non-conjugated BSA are given. The signal intensities obtained withthe 17-22, 18-23 and 19-24 peptides in relation to the 2-7 peptide arealso indicated. Peptide2-7 Peptide 17-22 Peptide 18-23 Peptide 19-24Peptide-ratio Peptide-ratio Peptide-ration MS-R # 2-7/BSA 17-22/BSA18-23/BSA 19-24/BSA 17-22/2-7 18-23/2-7 19-24/2-7 7 24 4 7 4 0.17 0.290.17 8 28 10 29 25 0.36 1.04 0.89 7.2 34 12 16 9 0.35 0.47 0.26 7.3 3411 15 9 0.32 0.44 0.26 7.4 36 10 13 6 0.28 0.36 0.17 7.9 28 9 13 8 0.320.46 0.29 7.11 37 11 15 9 0.30 0.41 0.24 7.12 38 6 8 7 0.16 0.21 0.188.1 30 1 11 8 0.03 0.37 0.27 8.2 32 4 28 23 0.13 0.88 0.72 3.2H2 26 1223 20 0.46 0.88 0.77 3.3H1 23 4 12 8 0.17 0.52 0.35 3.3H3 31 2 5 2 0.060.16 0.06 3.4H1 27 2 8 2 0.07 0.30 0.07 3.4H2 16 11 1 1 0.69 0.06 0.063.4H3 22 9 17 11 0.41 0.77 0.50 3.4H5 28 5 13 4 0.18 0.46 0.14 3.4H7 242 6 5 0.08 0.25 0.21 3.4H17 28 5 12 11 0.18 0.43 0.39 3.4L11 31 6 20 50.19 0.65 0.16 3.6H6 25 1 4 7 0.04 0.16 0.28 3.6H1 23 3 13 5 0.13 0.570.22 3.6H2 19 2 8 3 0.11 0.42 0.16 7.2H1 38 8 11 9 0.21 0.29 0.24 7.2H216 10 10 10 0.63 0.63 0.63 7.2H3 33 17 20 18 0.52 0.61 0.55 7.2H4 23 1213 12 0.52 0.57 0.52 7.2H5 30 13 18 15 0.43 0.60 0.50 7.2L1 24 14 16 110.57 0.68 0.45 7.4H1 31 16 20 16 0.52 0.65 0.51 7.4H2 36 17 20 16 0.470.56 0.46 7.9H1 32 7 12 6 0.23 0.36 0.19 7.9H2 35 3 6 8 0.08 0.16 0.237.9H3 35 11 20 9 0.31 0.57 0.27 7.9H4 30 10 15 7 0.32 0.49 0.22 7.11H131 8 9 8 0.25 0.29 0.25 7.11H2 34 10 12 14 0.29 0.36 0.41 7.12L1 16 1012 10 0.60 0.70 0.59 8.1H1 29 22 25 25 0.77 0.88 0.86 8.2H1 22 7 23 200.34 1.05 0.94 8.2L1 26 15 32 31 0.60 1.26 1.22

TABLE 7 Reactivity of MS-R IgGs and mouse monoclonal antibodies BAP-1,BAP-2, 4G8, 6E10 Amy-33 and 6F/3D with BSA-conjugated Aβ heptapeptides2-7 (AEFRHD, SEQ ID NO: 415), 17-22 (LVFFAE, SEQ ID NO: 420), 18-23(VFFAED, SEQ ID NO: 421) and 19-24 (FFAEDV, SEQ ID NO: 423). The ratiosof the ELISA read-out (optical density) obtained with peptide-conjugatedand non-conjugated BSA are given. The signal intensities obtained withthe 17-22, 18-23 and 19-24 peptides in relation to the 2-7 peptide arealso indicated. AEFRHD LVFFAE VFFAED FFAEDV (SEQ ID (SEQ ID No. (SEQ IDNo. (SEQ ID Peptide- Peptide- No. 415) 420) 421) No. 423) ratio ratioPeptide-ratio 2-7/BSA 17-22/BSA 18-23/BSA 19-24/BSA 17-22/2-7 18-23/2-719-24/2-7 MS-R IgG # 3.3 17 11 16 11 0.65 0.94 0.65 7.12 19 11 13 110.58 0.68 0.58 8.1 16 7 16 14 0.44 1.00 0.88 3.4H7 22 3 16 15 0.14 0.730.68 7.9H2 13 5 8 6 0.38 0.62 0.46 7.9H3 13 6 8 6 0.46 0.62 0.46 7.9.H730 5 16 10 0.17 0.53 0.33 7.11H2 10 6 7 6 0.60 0.70 0.60 8.2.H2 18 10 1514 0.56 0.83 0.78 3.6.H5 × 3.6.L2 11 7 9 8 0.64 0.82 0.73 7.11.H2 ×7.9.L1 14 8 10 9 0.57 0.71 0.64 (L1) 8.2.H2 × 8.2.L1 13 20 25 25 1.541.92 1.92 Mouse mab BAP-1 21 1 1 1 0.05 0.05 0.05 BAP-2 21 1 1 1 0.050.05 0.05 4G8 1 23 20 1 23 20 1 6E10 18 1 1 1 0.06 0.06 0.06 6F/3D* 1 11 1 1 1 1 Amy 33 16 2 1 3 0.13 0.06 0.19 *this antibody is specific forsequence 8-17 and does not recognize N-terminal or middle epitopesequences.

Example 13 Combination of Optimized H-CDR2 and L-CDR1 by Cross-Cloning

The modular design of the HuCAL library allows exchange ofcomplementarity determining regions (CDRs) of two different Fab encodinggenes in a simple cloning step. For a further improvement of affinitythe independently optimized H-CDR2 and L-CDR1 from matured clones withthe same H-CDR3 were combined, because there was a high probability thatthis combination would lead to a further gain of affinity (Yang et al.,1995, J. Mol. Biol. 254, 392-403; Schier et al., 1996b, J. Mol. Biol.263, 551-567; Chen et al., 1999, J. Mol. Biol. 293, 865-881). Wholelight chains, or fragments thereof, were transferred from an L-CDR1optimized donor clone to a H-CDR2 optimized recipient clone. Donor andrecipient clones were only combined, if both carried identical H-CDR3sequences. All donor and recipient clones carried the VH3-Vκ3 framework.

This was accomplished by transferring whole light chains from theL-CDR1-optimized donor clone to the H-CDR2-optimized recipient clone.Epitope specificity was conserved by only combining clones with the sameH-CDR3. By light chain exchange a H-CDR2-optimized clone obtained onlyan optimized L-CDR1, if the exchange occurred between clones with thesame L-CDR3. If the L-CDR3 of the clones to be combined was different,the H-CDR2-optimized clone acquired in addition to the optimized L-CDR1another L-CDR3 (L-CDR2 remained the HuCAL consensus sequence (Knappik etal., 2000)) and when derivatives of MS-Roche #7.12 were used as donorsof the light chain L-CDR1, 2 and 3 were exchanged in theH-CDR2-optimized acceptor clone. Three different cloning strategies wereemployed:

-   1) Using restriction endonucleases XbaI and SphI the whole antibody    light chain fragment was excised from plasmid 1 (e.g.    pMx9_Fab_MS-Roche#7.11.H1_FS) and the thereby obtained vector    backbone was then ligated to the light chain fragment of plasmid 2    (e.g. pMx9_Fab_MS-Roche#7.2.L1_FS) generated by XbaI and SphI    digest. Thereby a new plasmid (nomenclature:    pMx9_Fab_MS-Roche#7.11.H1×7.2.L1—FS) was created encoding L-CDR1,2,3    of parental clone #7.2.L1 and H-CDR1,2,3 of parental clone #7.11.H1.-   2) Using restriction endonucleases XbaI and Acc65I an L-CDR1 coding    fragment was excised from plasmid 1 (e.g.    pMx9_Fab_MS-Roche#7.11.H2—FS) and the thereby obtained vector    backbone was then ligated to the L-CDR1 fragment of plasmid 2 (e.g.    pMx9_Fab_MS-Roche#7.12.L1_FS) generated by XbaI and Acc65I. Thereby    a new plasmid (nomenclature:    pMx9_Fab_MS-Roche#7.11.H2×7.12.L1(L-CDR1)_FS) was created encoding    L-CDR1 of parental clone #7.12.L1 while L-CDR2,3 and H-CDR1,2,3 are    derived from parental clone #7.11.H2.-   3) Using restriction endonucleases XbaI and BamHI an L-CDR1 and    L-CDR2 coding fragment was excised from plasmid 1 (e.g.    pMx9_Fab_MS-Roche#7.11.H2—FS) and the thereby obtained vector    backbone was then ligated to the L-CDR1 and L-CDR2 fragment of    plasmid 2 (e.g. pMx9_Fab_MS-Roche#7.12.L1_FS) generated by XbaI and    BamHI digest. Thereby a new plasmid (nomenclature:    pMx9_Fab_MS-Roche#7.11.H2×7.12.L1(L-CDR1+2)_FS) was created encoding    L-CDR1 and L-CDR2 of parental clone #7.12.L1 while L-CDR3 and    H-CDR1,2,3 are derived from parental clone #7.11.H2.

Illustrative examples for the different cloning strategies as well asfor sequences donor and recipient clones are given in table 8.

After large scale expression and purification their affinities weredetermined on A8 (1-40) fibers. Furthermore, K_(D) values for selectedcross-cloned MS-R Fab/antibodies are given in appended Table 9.

TABLE 8 Binder name L-CDR1 pos.49 L-CDR2 pos. 85 L-CDR3 H-CDR1 pos.47H-CDR2 H-CDR3     cloning strategy 1)

MS-Roche #7.11.H1 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WGINAAGFRTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.2.L1 RASQYVDRTYLA YGASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYFDVMS-Roche #7.11.H1x7.2.L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS WGINAAGFRTYYADSVKG GKGNTHKPYGYVRYFDV     cloning strategy 2)

MS-Roche #7.11.H2 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WAINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.12.L1 RASQYVFRRYLA SGSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYFDVMS-Roche RASQYVFRRYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WAINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV #7.11.H2x7.12.L1(LCDR1)     cloningstrategy 3)

MS-Roche #7.11.H2 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS WAINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.12.L1 RASQYVFRRYLA SGSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYFDVMS-Roche RASQYVFRRYLA S GSSNRAT T QQVYSPPH GFTFSSYAMS WAINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV #7.11.H2x7.12.L1(LCDR1 + 2) MS-Roche#3.6H5 RASQSVSSSYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISESGKTKYYADSVKGLTHYARYYRVFDV MS-Roche #3.6L2 RASQFLSRYYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV MS-Roche #3.6H5x3.6L2RASQFLSRYYLA Y GASSRAT V QQTVNYPP GFTFSSYAMS W AISESGKTKYYADSVKGLTHYARYVRYFDV MS-Roche #3.6H8 RASQSVSSSYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISEYSKFKYYADSVKG LTHYARYYRYFDV MS-Roche #3.6L2RASQFLSRYYLA Y GASSRAT V QQTYNVPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRYFDV MS-Roche #3.6H8x3.6L2 RASQFLSRYYLA Y GASSRAT V QQTVNYPPGFTFSSYAMS W AISEYSKFKYYADSVKG LTHYARYYRYFDV MS-Roche #7.4.H2RASQSVSSSYLA Y GASSRAT V QQIYNFPH GFTFSSYAMS W AINYNGARIYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.2.L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPHGFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.4.H2x7.2.L1RASQYVDRTYLA Y GASSRAT T QQIYSFPM GFTFSSYAMS W AINYNGARIYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.9H2 RASQSVSSSYLA Y GASSRAT T LQIYNMPIGFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.12L2RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.9H2x7.12L2 RASQRFFYKYLA S GSSNRAT VLQLYNIPN GFTFSSYAMS W AINADGNRKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche#7.9H4 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINAVGMKKFYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.12.L2 RASQRFFYKYLA S GSSNRAT V LQLYNIPNGFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.9H4x7.12L2RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINAVGMKKFYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H1 RASQSVSSSYLA Y GASSRAT T QQVYSPPHGFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.11L1RASQRILRIYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W AISGSGGSTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H1x7.11L1 RASQRILRIYLA Y GASSRAT TQQVYSPPH GFTFSSYAMS W GINAAGFRTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche#7.11H1 RASQSVSSSYLA Y GASSRAT T QQVYSPPH GFTFSSYAMS W GINAAGFRTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.2L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPHGFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.11H1x7.2L1RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W GINAAGFRTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #3.3H1 RASQSVSSSYLA Y GASSRAT V HQMSSYPPGFTFSSYAMS W VISEKSRFIYYADSVKG LTHYARYYRYFDV MS-Roche #3.4L9RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRYFDV MS-Roche #3.3H1x3.4L9 RASRRIHVYYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISEKSRFIYYADSVKG LTHYARYYRYFDV MS-Roche #3.4H1RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETSIRKYYADSVKGLTHYARYYRYFDV MS-Roche #3.4L9 RASRRIHVYYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV MS-Roche #3.4H1x3.4L9RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISETSIRKYYADSVKGLTHYARYYRYFDV MS-Roche #3.4H3 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRYFDV MS-Roche #3.4L7RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRYFDV MS-Roche #3.4H3x3.4L7 RASQRLGRLYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISQTGRKIYYADSVKG LTHYARYYRYFDV MS-Roche #3.4H3RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADSVKGLTHYARYYRYFDV MS-Roche #3.4L9 RASRRIHVYYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV MS-Roche #3.4H3x3.4L9RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISQTGRKIYYADSVKGLTHYARYYRYFDV MS-Roche #3.4H7 RASQSVSSSYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRYFDV MS-Roche #3.4L9RASRRIHVYYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRYFDV MS-Roche #3.4H7x3.4L9 RASRRIHVYYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W VISETGKNIYYADSVKG LTHYARYYRYFDV MS-Roche #3.4H7RASQSVSSSYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADSVKGLTHYARYYRYFDV MS-Roche #3.4L7 RASQRLGRLYLA Y GASSRAT T QQTYDYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LTHYARYYRYFDV MS-Roche #3.4H7x3.4L7RASQRLGRLYLA Y GASSRAT T QQTYDYPP GFTFSSYAMS W VISETGKNIYYADSVKGLTHYARYYRYFDV MS-Roche #3.6H5 RASQSVSSSYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISESGKTKYYADSVKG LTHYARYYRYFDV MS-Roche #3.6L1RASQFIQRFYLA Y GASSRAT V QQTYNYPP GFTFSSYAMS W AISGSGGSTYYADSVKGLTHYARYYRYFDV MS-Roche #3.6H5x3.6L1 RASQFIQRFYLA Y GASSRAT V QQTYNYPPGFTFSSYAMS W AISESGKTKYYADSVKG LTHYARYYRYFDV MS-Roche #7.2H2RASQSVSSSYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINGTGMKKYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.2L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPHGFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.2H2x7.2L1RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINGTGMKKYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.4H2 RASQSVSSSYLA Y GASSRAT V QQIYNFPHGFTFSSYAMS W AINYNGARIYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.12L2RASQRFFYKYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.4H2x7.12L2 RASQRFFYKYLA S GSSNRAT VLQLYNIPN GFTFSSYAMS W AINYNGARIYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche#7.9H2 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINADGNRKYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.2L1 RASQYVDRTYLA Y GASSRAT T QQIYSFPHGFTFSSYAMS W AISGSGGSTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.9H2x7.2L1RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AINADGNRKYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H2 RASQSVSSSYLA Y GASSRAT T QQVYSPPHGFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.2L1RASQYVDRTYLA Y GASSRAT T QQIYSFPH GFTFSSYAMS W AISGSGGSTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H2x7.2L1 RASQYVDRTYLA Y GASSRAT TQQIYSFPH GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche#7.9H2 RASQSVSSSYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AINADGNRKYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.12L1 RASQYVFRRYLA S GSSNRAT V LQLYNIPNGFTFSSYGMS W NISGSGSSTYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.9H2x7.12L1RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYAMS W AINADGNRKYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H2 RASQSVSSSYLA Y GASSRAT T QQVYSPPHGFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.9L1RASQRLSPRYLA Y GASSRAT T LQIYNMPI GFTFSSYAMS W AISGSGGSTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H2x7.9L1 RASQRLSPRYLA Y GASSRAT TLQIYNMPI GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche#8.1H1 RASQSVSSSYLA Y GASSRAT T QQLSNYPP GFTFSSYAMS W AISRSGSNIYYADSVKGLLSRGYNGYYHKFDV MS-Roche #8.2L1 RASQRVSGRYLA Y GASSRAT T QQLSSYPPGFTFSSYAMS W AISGSGGSTYYADSVKG LLSRGYNGYYHKFDV MS-Roche #8.1H1x8.2L1RASQRVSGRYLA Y GASSRAT T QQLSSYPP GFTFSSYAMS W AISRSGSNIYYADSVKGLLSRGYNGYYHKFDV MS-Roche #7.11H2 RASQSVSSSYLA Y GASSRAT T QQVYSPPHGFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV MS-Roche #7.12L1RASQYVFRRYLA S GSSNRAT V LQLYNIPN GFTFSSYGMS W NISGSGSSTYYADSVKGGKGNTHKPYGYVRYFDV MS-Roche #7.11H2x7.12L1 RASQYVFRRYLA S GSSNRAT VLQLYNIPN GFTFSSYAMS W AINANGYKKYYADSVKG GKGNTHKPYGYVRYFDV Arrowsindicate the location of restriction enzyme sites used to digestcorresponding plasmids

TABLE 9 K_(D) values for crosscloned MS-R Fab binding to Aβ1-40 andAβ1-42 fibers as determined in the Biacore. The preparation ofcrosscloned Fabs is described in example 13. The K_(D) values weredetermined by kinetic curve fittings and corrected for the content ofactive Fab present in each sample as described in the text. Some of theFabs were additionally purified by size exclusion chromatography orpreparative ultracentrifugation to remove aggregated material. (L1), theH-CDR2- matured acceptor clone received only L-CDR1 from the L-CDR1improved donor clone; (L1 + 2), the H-CDR2-matured acceptor clonereceived L-CDR1 + 2 from the L-CDR1 improved donor clone. K_(D) Aβ₁₋₄₀K_(D) Aβ₁₋₄₂ MS-R # nM nM 3.3H1 × 3.4L9 2.16 2.97 3.4H1 × 3.4L9 0.25 0.53.4H3 × 3.4L7 0.92 0.92 3.4H3 × 3.4L9 1.05 0.93 3.4H7 × 3.4L9 2.66 3.513.4H7 × 3.4L7 1.19 1.23 3.6H5 × 3.6L1 1.25 1.04 3.6H5 × 3.6L2 1.26 0.847.2H2 × 7.2L1 1.29 1.43 7.4H2 × 7.2L1 1.4 1.4 7.4H2 × 7.12L2 1.4 1.87.9H2 × 7.2L1(L1) 1.4 1.4 7.9H2 × 7.12L1 1.2 1.1 7.9H2 × 7.12L2(L1 + 2)0.4 0.4 7.11H1 × 7.2L1 1.75 1.39 7.11H1 × 7.11L1 0.41 0.47 7.11H2 ×7.2L1(L1) 1 0.6 7.11H2 × 7.9L1 (L1) 0.1 1 8.1H1 × 8.2L1 1.3 1.6

Example 14 In Vivo Amyloid Plaque Decoration in a Mouse Model ofAlzheimer's Disease as Revealed by Confocal Laser Scanning Microscopyand Colocalization Analysis

Selected MS-R IgG1 antibodies were tested in APP/PS2 double transgenicmice (Reference: Richards et al., Soc. Neurosci. Abstr., Vol. 27,Program No. 5467, 2001) for amyloid plaque decoration in vivo. Theantibodies (1 mg/mouse) were administered i.v. and after 3 days thebrains were perfused with saline and prepared for cryosection. Inanother study the mice were exposed to higher concentrations of theantibodies, i.e. 2 mg injected i.v. at day 0, 3, and 6, and sacrificedat day nine. The presence of the antibodies bound to amyloid plaques wasassessed on unfixed cryostat sections by double-labeled indirectimmunofluorescence using goat anti-human IgG (H+L) conjugated to eitherCy3 (#109-165-003, Jackson Immuno Research) followed by BAP-2-Alexa488immunoconjugate. Imaging was done by confocal laser microscopy and imageprocessing for quantitative detection of colocalizations by IMARIS andCOLOCALIZATION software (Bitplane, Switzerland). Typical examples areshown in FIGS. 10-14. All of the MS-R antibodies tested were foundpositive in immunodecoration of amyloid plaques in vivo, although somevariability was noted.

Example 15 Investigation of Binding of Different Monoclonal Antibodiesto Amyloid Precursor Protein (APP) on the Surface of HEK293 Cells

APP is widely expressed in the central nervous system. Binding ofantibody to cell surface APP may lead to complement activation and celldestruction in healthy brain areas. Therefore, it is mandatory fortherapeutic A-beta antibodies to be devoid of reactivity towards APP.High affinity antibodies against the N-terminal domain of A-beta (e.g.BAP-1, BAP-2) recognize the respective epitope also in the framework ofAPP. In contrast, the antibodies against the middle epitope (e.g. 4G8),and the antibodies of the invention are surprisingly unable to recognizeto cell surface APP. Thus, antibodies of the invention which decorateA-beta, but not APP in vivo, are superior to non-selective antibodies.

The method of flow cytometry is well known in the art. Relative units offluorescence (FL1-H) measured by flow cytometry indicate cell surfacebinding of the respective antibody. A fluorescence shift on APPtransfected HEK293 compared to untransfected HEK293 cells indicates theunwanted reaction with cell surface APP. As an example, antibodies BAP-1and BAP-2 against the N-terminal domain show a significant shift of FL-1signal in HEK293/APP (thick line) compared to untransfected HEK293 cells(dotted line). The 4G8 antibody (specific for the middle A-beta epitope)and all antibodies of the invention (specific for N-terminal and middleA-beta epitopes) show no significant shift in fluorescence. Differencesin basal fluorescence between HE293/APP ad HEK293 cells are due todifferent cell size. A FACScan instrument was used in combination withthe Cellquest Pro Software package (both Becton Dickinson).

Example 16 List of Identified SEQ ID NOs Relating to Inventive AntibodyMolecules

The appended table 10 relates to sequences as defined herein for somespecific inventive antibody molecules.

TABLE 10 Identification of SEQ ID NOs for parental antibodies as well asoptimized, matured and/or cross-cloned antibody molecules HCDR3 HCDR3LCDR3 LCDR3 Molecule # VH prot VL prot VH DNA VL DNA prot DNA prot DNA 34 10 3 9 22 21 16 15 7 6 12 5 11 24 23 18 17 8 8 14 7 13 26 25 20 193.6H5 × 3.6L2 33 47 32 46 61 60 75 74 3.6H8 × 3.6L2 35 49 34 48 63 62 7776 7.4H2 × 7.2L1 37 51 36 50 65 64 79 78 7.9H2 × 7.12L2 39 53 38 52 6766 81 80 7.9H4 × 7.12L2 41 55 40 54 69 68 83 82 7.11H1 × 7.11L1 43 57 4256 71 70 85 84 7.11H1 × 7.2L1 45 59 44 58 73 72 87 86 7.9H7 89 91 88 9093 92 95 94 3.3H1 × 3.4L9 295 325 294 324 355 354 385 384 3.4H1 × 3.4L9297 327 296 326 357 356 387 386 3.4H3 × 3.4L7 299 329 298 328 359 358389 388 3.4H3 × 3.4L9 301 331 300 330 361 360 391 390 3.4H7 × 3.4L9 303333 302 332 363 362 393 392 3.4H7 × 3.4L7 305 335 304 334 365 364 395394 3.6H5 × 3.6L1 307 337 306 336 367 366 397 396 7.2H2 × 7.2L1 309 339308 338 369 368 399 398 7.4H2 × 7.12L2 311 341 310 340 371 370 401 4007.9H2 × 7.2L1 313 343 312 342 373 372 403 402 7.9H2 × 7.12L1 315 345 314344 375 374 405 404 7.11H2 × 7.2L1 317 347 316 346 377 376 407 4067.11H2 × 7.9L1 319 349 318 348 379 378 409 408 7.11H2 × 7.12L1 321 351320 350 381 380 411 410 8.1H1 × 8.2L1 323 353 322 352 383 382 413 412

1. An antibody molecule capable of specifically recognizing two regionsof the β-A4 peptide/Aβ4, wherein the first region comprises the aminoacid sequence AEFRHDSGY as shown in SEQ ID NO: 1 or a fragment thereofand wherein the second region comprises the amino acid sequenceVHHQKLVFFAEDVG as shown in SEQ ID NO: 2 or a fragment thereof, whereinsaid antibody molecule comprises (a) a variable V_(L)-Region comprisingcomplementary determining regions, L-CDR1, L-CDR2, and L-CDR3, wherein:(1) L-CDR1 comprises a sequence selected from the group consisting ofSEQ ID NOs: 96, 160, 175-177, 180, 189-190, 200-201, and 206-210; (2)L-CDR2 comprises a sequence selected from the group consisting of SEQ IDNOs: 97 and 161; and (3) L-CDR3 comprises a sequence selected from thegroup consisting of SEQ ID NOs: 18, 79, 81, 95, 149, 151-156, 158-159and 166; and (b) a variable V_(H)-Region comprising complementarydetermining regions, H-CDR1, H-CDR2, and H-CDR3, wherein: (1) H-CDR1comprises a sequence selected from the group consisting of SEQ ID NOs:99 and 163; (2) H-CDR2 comprises a sequence selected from the groupconsisting of SEQ ID NOs: 100, 164, 167-169, 170-174, 179, 181-182,184-188, 192-197, 199 and 204; and (3) H-CDR3 comprises SEQ ID NO: 24.2. The antibody molecule of claim 1, wherein said antibody moleculerecognizes at least two consecutive amino acids within the two regionsof β-A4.
 3. The antibody molecule of claim 1, wherein said antibodymolecule recognizes in the first region an amino acid sequence selectedfrom the group consisting of EF, EFR, FR, and SEQ ID NOs: 415-418, andin the second region an amino acid sequence selected from the groupconsisting of LV and SEQ ID NOs: 419-423.
 4. The antibody molecule ofclaim 1, wherein said antibody molecule comprises a variableV_(H)-region comprising a sequence selected from the group consisting ofSEQ ID NOs: 4, 37, 39, 41, 43, 89, and
 425. 5. The antibody molecule ofclaim 1, wherein said antibody molecule comprises a variableV_(L)-region comprising a sequence selected from the group consisting ofSEQ ID NOs: 12, 51, 53, 57, and
 91. 6. (canceled)
 7. The antibodymolecule of claim 1, wherein said antibody is selected from the groupconsisting of MSR 7 and an affinity-matured version of MSR
 7. 8. Theantibody molecule of claim 1, wherein said antibody molecule is a fullantibody (immunoglobulin), a F(ab)-fragment, a F(ab)₂-fragment, asingle-chain antibody, a chimeric antibody, a CDR-grafted antibody, abivalent antibody-construct, a synthetic antibody or a cross-clonedantibody.
 9. The antibody molecule of claim 1, wherein two regions ofβ-A4 form a conformational epitope or a discontinuous epitope. 10.(canceled)
 11. A nucleic acid molecule encoding an antibody moleculeaccording to claim
 1. 12. A vector comprising the nucleic acid moleculeof claim
 11. 13. A host cell comprising the vector of claim
 12. 14. Amethod for the preparation of an antibody molecule comprising culturingthe host cell of claim 13 under conditions that allow synthesis of saidantibody molecule and recovering said antibody molecule from saidculture.
 15. A pharmaceutical or diagnostic composition comprising anantibody molecule according to claim 1 and a carrier or diluent.
 16. Thecomposition of claim 15, which is a pharmaceutical composition. 17-21.(canceled)
 22. A kit comprising an antibody molecule according to claim1, a nucleic acid molecule according to claim 11, a vector according toclaim 12, or a host cell according to claim 13, wherein the antibody,nucleic acid, vector or host cell is contained in at least one vial,bottle, container or multicontainer unit. 23-28. (canceled)
 29. Acomposition comprising an antibody molecule produced by the method ofclaim
 14. 30. The composition of claim 16 further comprising apharmaceutically acceptable carrier and/or diluent.